Robert F. Campbell and Melvin W. Hanna
1892
The Vanadyl Ion as an Electron Paramagnetic Resonance Probe of Micelle-Liquid Crystal Systems Robert
F. Campbell*
and Melvin W. Hanna
Department of Chemistry, University of Colorado, Boulder, Colorado 80302 (Received April 27, 1976)
Various phases of the three-component micelle-liquid crystal system, sodium octanoateloctanoic acid/ water, were “spin labeled” with the paramagnetic vanadyl ion, V02+. These phases contained such multimolecular structures as water-internal and water-external micelles, water-internal and water-external rodshaped liquid crystals and lamellar liquid crystals. The EPR spectrum of the vanadyl ion bound to these structures was found to reflect molecular motion of the polar interface of these structures. Further, the spectra indicated that motion in nearly all structures was on the same timescale. A qualitative estimate of this timescale was made. A quantitative determination of this timescale (which may have pointed up small differences in this timescale from one phase to another) was not possible, for the EPR spectrum of the vanadyl ion bound to these structures was of the slow-motional type, and existing relaxation theories are not applicable in this case. However, it was postulated that the newly developed relaxation theory, the stochastic Liouville method, applied to the vanadyl spectrum would offer the solution to this problem.
I. Introduction It is becoming increasingly apparent that the paramagnetic labeling technique of EPR spectroscopy is a powerful technique for probing the structure and dynamics of macromolecular systems. Investigations employing nitroxide spin labels incorporated in biological mernbranes,l oriented nematic and smectic liquid crystal^,^-^ and micellar solutions5 have provided both dynamic information on the incorporated probe,< as well as structural information on the macromolecular species itself. More recently, the technique has been extended to the use of paramagnetic metal ions as “spin probes”. Due to its relatively uncomplicated magnetic interactions, the vanadyl ion, V02+, is fast becoming a most useful member of this class of probe. It has been successfully employed to gain structural information on metal ion binding sites in proteins? to report on the motion at the anionic vesicle surface of a phospholipid bilayer7 and to query counterion mobility in micellar solutions.8Also, the vanadyl acetylacetonate complex has been employed as a probe of the long-ranged ordering occurring in nematic liquid cry~tals.~-ll For some time, the internal structure of the molecular aggregates of micelle-liquid crystal systems has been of interand is now generally understood. However, much less progress has been made in investigating the polar interfacial region of such aggregates. We felt that the vanadyl probe might provide a direct method for investigating this region, and here we report on its use as a probe of such systems. 11. Experimental Section A. EPR Spectrometer. All EPR measurements were made with a Varian V 4502 spectrometer operating a X-band (9.5 GHz) with 100-kHz field modulation in conjunction with a Varian rectangular cavity. The temperature was not controlled, but never varied more than fl “C as measured at the center of the cavity with a calibrated thermometer. Measurements were taken at the beginning and end of each experiment. The temperature between experiments never varied
* Correspondence should be addressed to this author at the Department of Chemistry, Cornel1University, Ithica, N.Y. 14853. The Journal of Physical Chemistry, Vol. 80, No. 17, 1976
more than f 2 “C about a mean ambient temperature of 22 “C. The room temperature spectra were recorded using an aqueous quartz flat cell; however, to determine if any longrange ordering of the sample resulted from the use of the flat cell, comparison spectra were run using 2-mm i.d. quartz tubes. The 77 K spectra were recorded using a quartz EPR dewar-liquid nitrogen arrangement. The cavity frequency changed from 9.53 GHz with the flat cell to 9.09 GHz with the quartz dewar. The microwave frequency was measured with a Micro-Now Model 101 frequency multiplier in conjunction with a Hewlett-Packard 5245M frequency counter. The magnetic field was measured with a proton probe in conjunction with the Hewlett-Packard frequency counter. The spectra were recorded on a Honeywell x-t strip chart recorder equipped with a field marker. Effects of overmodulation and saturation on the line shape were avoided. B. Determination of the Rigid Limit Magnetic Tensor Components. All rigid limit parameters, i.e., All, Al, and gll, g, were determined from 77 K frozen solution samples. All samples froze into amorphous solids when either slowly or rapidly emersed in liquid Nz.In order to accurately determine these parameters, one must be able to pick out the “principal resonance fields”. This is somewhat difficult with vanadyl spectra for the central portion is complicated by overlap of the parallel and perpendicular resonance fields. In general, the principal resonance fields cannot be determined by inspection, and computer simulation of the spectrum is required. All rigid limit parameters, therefore, were verified by computer simulation. The method of Chasteen6 was employed for this purpose. C. Sample Preparation. Vanadyl Stock Solution. A I M stock solution of the vanadyl probe was prepared by dilution of vanadyl sulfate (Alfa Inorganics) with doubly distilled water and subsequent adjustment of the pH to 2.0 by slow addition of sodium hydroxide. To avoid oxidation of the vanadyl ion, the solution was deoxygenated by bubbling with moist, prepurified N2 gas during, and at least 1h before, the addition o f the sodium hydroxide, The solution was standardized spectrophotometrically at 750 ram using a molar extinction
V02+ Probe of Micelle-Liquid Crystal Systems
coefficient of 18.0(f0.2).15The solution was stored a t 4 "C under N2 and, over a 6 month period, the vanadyl concentration decreased less than 1%. Sodium Octanoate. Sodium octanoate was prepared by stoichiometric reaction of octanoic acid (Golden Label, 99.5%, Eastman Kodak) with sodium hydroxide (Mallenkrodt, reagent) in absolute ethanol. The resulting product was washed with absolute ethanol, filtered, and oven dried at 65-75 "C for 24 h. The finely ground product was then placed for 24 h in an oil pump evacuated bell jar containing CaClz to ensure an anhydrous product. The yield was about 75%. The product was checked for acid-base impurity by titration with perchloric acid in glacial acetic acid solvent, using crystal violet indicator. A potentiometric verification of the crystal violet endpoint was made, and titration of anhydrous sodium acetate (Mallendrodt, reagent) gave a further check on it. This check gave a molecular weight for the sodium acetate of 82.07(f0.03);actual mol wt = 82.04. Titration of the sodium octanoate gave a molecular weight of 166.6 (f0.2);actual mol wt = 166.2. Micelle-Liquid Crystal Phases and V02+Labeling. Appropriate amounts of sodium octanoate and octanoic acid (Golden Label, 99.5%, Eastman Kodak) were weighed out in 5-dram Kimble vials (27.25 X 55 mm) containing a 10-mm teflon coated magnetic stirring bar. The vial was immediately serum capped and the appropriate weight of doubly distilled water added via a previously calibrated glass syringe operated with a Cornwall plunger-type dispenser. Samples were always prepared based upon the same weight of water so that the syringe need only be calibrated once. The solutions were then deoxygenated by bubbling with moist, prepurified Nz for 1h.l6 To ensure that the solutions had reached equilibrium, they were slowly magnetically stirred under a positive pressure of N2 for 24 h. Some of the liquid crystalline samples were too viscous to be deoxygenated by N2 bubbling. These samples were prepared by purging, with N2, the serum capped vials containing sodium octanoate and octanoic acid, and then syringing in previously deoxygenated doubly distilled water. The resulting mixture was then heated to above the liquid crystal to isotropic liquid transition temperature (-160 "C) and allowed to equilibrate at room temperature under N2 for 24 h. The vanadyl probe was always added such that its final concentration was low enough that there was no exchange broadening. Thus the concentration of the probe was always less than or equal to 1 X 10-3 M. The EPR spectra of the samples were recorded in previously N2 purged, serum capped sample cells. The sample was introduced via a Nz flushed syringe. The very viscous liquid crystalline samples had to be heated in order to be transferred. In such cases, the heated isotropic liquid was drawn into the syringe and then allowed to equilibrate to room temperature before transfer to the EPR sample cell. Short term heating for 5 min or less caused little oxidation of the vanadyl probe.
111. The Micelle-Liquid Crystal System For our investigation, we chose the sodium octanoateloctanoic acid/water system. A survey of the literature17 shows other systems are available, however, this system manifests two desirable qualities. First, the vanadyl ion forms stable complexes with carboxylates.ls Second, this system has been fairly well c h a r a ~ t e r i z e d l ~and - ~ ~provides several different single phase regions of molecular organization for study. The essential features of the system are given in the three component phase diagram,23 Figure 1. It consists, using Ek-
1893
"IoOctanoic acid
10
20
30
40
50
60
70 80
SX3
Figure 1. Three-component phase diagram of the sodium octanoatel octanoic acidlwater system at 20 "C: L,, water-exterpal micellar solution; Lp, water-internal micellar solution; B-F, liquid crystalline phases. (% = percent by weight.)
wall's notation,l' of two micellar phases, L1 and L2, and five mesomorphous (smectic liquid crystalline) phases, B-F. Reportedly,20 phases B, C, and D have a lamellar (molecular bileaflet) structure, while phase E and F possess hexagonally packed, water-external and water-internal rod-shaped structures, respectively. The phase L1 consists of water-external micelles with and without solubilized octanoic acid. Phase L2 consists of molecular complexes of sodium octanoate and octanoic acid, up to approximately 20% water, waterinternal micelles up to about 40% water, and water-external micelles beyond. The various structures are represented in Figure 2.
IV. Results and Discussion The room temperature and frozen solution spectra24of the vanadyl probe added to each of the composition points labeled a-h in Figure 1 are given in Figures 3 and 4. The exact composition of each of the points, along with their macroscopic viscosities and pH, is given in Table I. As can be seen, the spectra are generally similar in appearance. Both display highly asymmetric line shapes and satellite peaks at the wings. The room temperature resonances, however, are considerably broadened and shifted relative to those of the frozen soluti0n.~5Such results indicate that the vanadyl probe is undergoing slow molecular reorientation at room temperature; molecular reorientation being quenched at 77 K. Slow reorientation would result if the probe were bound firmly to the large molecular aggregates under investigation, and these spectra encourage this conclusion. However, in solutions of high macroscopic viscosity, reorientation of the unbound probe would also be slow. Thus, the first question that must be answered is whether the similar slow motional spectra arise from the fact that V02+ is bound to the molecular aggregates in each phase, or whether they are due to slow tumbling of the V02+ ion in viscous solution. Only when the probe is bound to the aggregates, will it report directly on them. A. Binding Evidence. We have found that slow motional spectra occur when the vanadyl probe is added to glycerine solutions of 7 > 15 cP. Consulting Table I, one sees that, except for point f, the macroscopic viscosity of all composition points exceed this limit, thus, the observation of a slow motional spectrum at these points does not provide absolute evidence that the probe is bound. There is, however, considerable overlap in evidence supporting a bound probe. The Journal of Physical Chemistry, Vol. 80, No. 17, 1976
1894
Robert F. Campbell and Melvin W. Hanna
1353
3430
2833
F1E.C !GI
Figure 2. Micellar and liquid crystalline structures of the sodium octanoate/octanoic acid/water system. I
I :
I
Figure 3. X-band (9.5 GHz) room temperature and 77 K frozqn solution EPR spectra of composition points, a-h. (A) Points e (lamellar region) and g (H20-externalmicelles)gave the solid.line spectrum. Points b and f (H20-external micelles) gave the dashed line spectrum. Point a (H20-internal micelles) gave the dotted line spectrum. (B) Point c (H20-internalrods) gave the solid line spectrum, while point h (H20external rods) gave the dotted line spectrum. (C) A representative 77 K frozen solution spectrum of points a-h. The first three high field parallel 51V nuclear hyperfine lines labeled MI = +7/2, +5/2, +3/2 are indicated. First, if the vanadyl ion binds to the molecular aggregates under investigation, it will do so a t their polar interface (i.e., at the carboxyl groups of the fatty acid constituents). We have determined the rigid limit EPR parameters from the 77 K frozen solution spectrum of each composition point, as well as from the 77 K frozen solution of vanadyl acetate. Consulting Table 11, we see that these parameters are the same, within experimental uncertainty, as those of the acetate complex. Since these parameters are sensitive to changes in composition,6,26they indicate that not only is a vanadyl carboxylate formed in each of the regions investigated but, further, that it has the same coordination in each region as well. Second, the observation of a slow motional spectrum a t composition point f, where the viscosity is 2.2 cP, confirms that the probe is bound to the micelles of this region. In a solution of macroscopic viscosity -2 cP, an unbound probe would be free to undergo rapid isotropic reorientation, and therefore would give a characteristic eight-line (Le., rapid motional) The Journal of Physical Chemistry, Vol. 80, No. 17, 1976
Flgure 4. X-band (9.5 GHz) room temperature EPR spectra showing preferential orientation in the lamellar mesophase D, point d. The solid and dashed line spectrum represent the perpendicular and parallel hyperfine lines, respectively. The solid line spectrum shows the enhancement of the perpendicular hyperfine lines obtained with the EPR flat cell normal perpendicular to the Ho direction. The dashed line spectrum shows the enhancement of the parallel hyperfine lines obtained with the flat cell normal parallel to Ho. spectrum. Moreover, as will be discussed, investigation of region D, point d shows the spectrum to be due to the preferential orientation of the probe in this lamellar mesophase. Here too, the probe must be bound; if it were not, it would be reorienting rapidly in the water layers which separate these molecular bilayers from one another.28 Third, we have investigated the stability of the vanadyl acetate and micelle complex (7N 2 cP)in the pH range from 10 to 5 and found that their stability increases with decreasing pH. The vanadyl acetate rapid motional spectrum becomes detectable and grows in intensity from pH 7.9 downward. Owing to the larger overall negative charge of the micelle, the vanadyl-micelle complex slow motional spectrum becomes observable and increases in intensity from pH 8.4 downward. Based upon the pH data of Table I, it is probable that the vanadyl probe binds in each of the regions investigated. When taken together, the above evidence strongly supports our conclusion that the probe is bound to the polar heads of the molecular aggregates at each composition point investigated. B. Molecular Motion. The room temperature spectra all look very much the same and exhibit resonance lines which are asymmetric, broadened, and shifted from the rigid limit. Polnaszek, Bruno, and Freed (PBF) have shown that these are the spectral characteristics exhibited by a paramagnetic probe undergoing slow motion in both isotropicz9and anisotropic30 liquids. In such cases, the effect of motion, from the rigid limit, manifests itself in a decrease in the observed A 1 , i.e., A /I*,and an increase in the observed A I, Le., A 1.*, as well as in changes in the relative intensities of the parallel and perpendicular resonances due to line broadening. Thus, the wings of the slow motional spectrum move inward from their rigid limit positions as motion increases. Such is the behavior displayed by our room temperature spectra. Comparison of the rigid limit and slow motional spectra of the vanadyl probe (Figure 3) shows that there are at least two pairs of hyperfine extrema, i.e., wings, corresponding to the M I = f 7 / 2 and f 5 / 2 parallel resonances which are well resolved in the room temperature spectra. Freed3l has shown that the parameter S = Ai */Ail, where All* is the separation between a corresponding pair of slow motional hyperfine extrema and All is the rigid
V02+ Probe of Micelle-Liquid Crystal Systems
189s
TABLE I: Data on Composition Points
Viscosity (20 "C), CP Lz L2 F D B L1
a C
d e f g h
L1
E a
50.02 27.97 46.03 32.00 9.94 1.94 7.07 7.00
20.01 12.02 40.97 30.01 10.02 10.02 34.99 42.00
b
62.4c 36.OC
29.97 60.01 12.99 37.99 80.04 88.04 57.94 51.00
d d d
2.2" 78" d
pH (f0.l) 5.6 5.9 6.7 6.1 6.7 7.3 7.5 7.6
Sodium octanoate. Octanoic acid. Reference 21. Immeasurable because of its gel-like character. e Estimated error of 10%.
TABLE 11: Anisotropic ESR Parameters of Points a-h
Point
gl; (f0.002)
a b
1.938 1.940 1.940 1.940 1.940 1.942 1.942 1.940 1.941
C
d e f
g h Vanadyl acetate
g l (fO.001) go (f0.002)a Ai1(10-~ cm-l) (f1.7)
1.977 1.976 1.976 1.976 1.976 1.976 1.976 1.976 1.976
+
Computed from go = l/3(glI 2gl).
1.964 1.964 1.964 1.964 1.964 1.965 1.965 1.964 1.964
99.7 97.7 97.6 98.9 98.8 96.9 96.5 97.7 98.3
+
Region
Point
Au.G
Ail*. G
S = Au*/Au
Lz Lz F D B L1 L1 E
a b
192(f2) 190(f2) 190(f2) 192(f2) 192(f2) 188(f2) 188(f2) 190(f2)
170(f3) 164(f3) 180(f3) 170(f3) 167(f3) 163(f4) 168(f3) 167(f3)
0.89(f0.03) 0.86(f0.03) 0.95(f0.03) 0.89( f0.03) 0.87(f0.03) 0.87(f0.03) 0.89(&0.03) O.SS(f0.03)
d e f g h
62.7 60.6 60.7 61.5 61.5 59.9 59.5 60.6 61.1
Computed from A. = '$,(AI] 2AJ.
TABLE 111: S Parameters
C
173.8 172.0 172.1 173.8 173.5 170.8 170.6 172.0 172.8
A1(10-4 cm-l) (fl.O) Ao(lOU4cm-l)b (f1.2)
limit value of this quantity, is proportional to the correlation time, 7c, characterizing the molecular motion. As the value of S increases to the value 1, the molecular motion becomes slower, finally reaching the rigid limit at 1.Thus, S is a qualitative measure of the molecular motion. With the application of the methods of PBF,29,30the value of 7c could be quantitatively determined. Values of S computed for each of the composition points investigated are given in Table 111. Comparing these values, we see that all are the same within experimental uncertainty, except region F, point c. Comparison of the room temperature spectrum of region F with those of the other regions clearly shows slower motion in this region; even the M I = f 3 / 2 parallel hyperfine lines are partially resolved. Given these values of S, it seems that the molecular motion is on the same timescale in each of the regions investigated, except possibly region F. Unfortunately, without a detailed mathematical analysis of the vanadyl slow motional spectra via the methods of PBF, a quantitative determination of T~ cannot be made. This is currently beyond the scope of this work. On a qualitative level, however, some progress can be made. First, slow motional spectra are observed when the rotational frequency of the paramagnetic molecule, 1 / ~is~slow , com-
pared to the hyperfine anisotropy, I A 11 - A I in Hz. For nitroxide radicals, the slow motional region has been shown to s,32 the hyperfine ans < 7c < be characterized by isotropy being -10s Hz. For vanadyl, this anisotropy is -lo9 Hz, and therefore the slow motional region sets in at values of 7c an order of magnitude smaller, that is, a t 7c 10-9 s. Thus, the timescale for the motion observed in our system must be greater than this value. Second, we have investigated the slow motional spectrum of the aqueous vanadyl ion at low pH33 and varying viscosity in glycerine. We found that slow motional spectra, very similar to those of the micelle-liquid crystal system, occur in the viscosity range 100-300 cP. Since the hydrodynamic radius of aqueous vanadyl has been determined,ls an estimate of 7c for such spectra can be made using the Stoke's law relation:
-
7c =
4lIr37/3kT
(1)
We find that these spectra correspond to 4 X s 5 T~ 5 1 x 10-8 s. Up to this point, we have not discussed the source of the motion being monitored by the vanadyl probe. Since the probe is bound to the various structures investigated, it reports directly on their motional properties. The motion could be of two obvious types: (1)overall reorientation of the molecular aggregates, or (2) local motion at the polar interface of the aggregates; the probe being bound at the carboxyl groups of these structures. The first type of motion can be ruled out. If the EPR spectra were determined by the overall reorientation of the molecular aggregates, one would expect significant differences in the spectra due to the labeled liquid crystals and those due to the labeled micelles. The lamellar liquid crystals would undergo essentially no overall reorientation, since these structures exist as large bileaflet sheets. Therefore, this region would give a nearly rigid limit spectrum. The rod-shaped liquid crystals would, at most, undergo slow reorientation compared to the micelles, since they exist as long rods. In diThe Journal of Physical Chemistry, Vol. 80, No. 17, 1976
1896
lute micelle regions, such as region L1, point f, the overall reorientation would be much faster. Using the Stoke’s law relation, one can estimate the value of the rc for overall reorientation in this region. Here, the micelles are reportedly spherical,lg having a radius of about 27 A,34 and for the vanadyl probe bound firmly to the surface of a rigid, spherical micelle in water the Stoke’s law relations gives, T~ = 2 X 10-8 s.35This value implies that the dilute micelle spectrum could be the result of overall reorientation. However, given the spectral similarity of all the other regions investigated this seems inconsistent. We conclude that the vanadyl probe reports on the local motion at the polar interface of all the macromolecular structures investigated. C. Structural Information. Of the regions investigated, only region D produced any structure i n f ~ r m a t i o n The . ~ ~ spectrum of this region, point d, shows a unique feature. . . preferential orientation of the molecular bilayers. Figure 4 shows that with the EPR flat cell normal perpendicular to the externally applied magnetic field, Ho, enhancement of the perpendicular resonance fields occurs. In addition with the flat cell normal parallel to Ho, enhancement of the parallel resonance fields occurs. This phenomenon is consistent with the results of nitroxide labeling experiments in a similar system.z These experiments showed that macroscopic ordering of this type of liquid crystal, Le., smectic phase D, occurs when it is forced between two closely spaced parallel plates such as those of the EPR flat cell. Shearing forces and contact with the plate surfaces align the molecular bilayers parallel to the plate surfaces?’ Such orientation behavior is consistent with and supports the past structural assignment of lamellarz0 to this smectic phase. In addition to structural verification of this smectic phase, the basic coordination geometry of the vanadyl probe, bound to the molecular bilayers, can be determined. Since the vanadyl magnetic interactions are well described by axially symmetric g and A tensors, one invariably finds that the g shift and hyperfine splittings are greatest when the externally applied magnetic field is directed along the V=O bond direction. With the V=O bond parallel to No,one obtains the “parallel” spectrum; this has the largest hyperfine splittings. With the V=O bond perpendicular to Ho, one obtains the “perpendicular” spectrum; this has the smallest hyperfine splittings. Thus, the V=O bond direction in this smectic phase is readily determined by orienting the bilayer with respect to the applied magnetic field until the maximum and minimum hyperfine splittings are obtained. Such spectra are shown in Figure 4. These spectra show that (1)the V=O bond direction is essentially perpendicular to the bilayer surface. The contributions from the perpendicular spectrum in the parallel spectrum, and vice versa, are attributed to some misalignment of the bilayers due to orientation at the edges of the flat cell and to inclusion of air bubbles in the (2) Ail* and A l * display behavior consistent with a slow motional spectrum; that is, All* < All and A L * > A l . The results of PBF for anisotropic liquids30 with a high degree of order substantiate such behavior. Further, the fact that S for this region is the same as for the other regions, where slow motional effects are clearly observed, confirms that the spectra from this ordered region exhibit the effects of slow motion. Thus, we conclude that the probe “sits down” with its V=O bond perpendicular to the polar interface of the bilayer and undergoes slow motion. The consistency of the rigid limit EPR parameters for each region indicates that the vanadyl complex formed is identical. Therefore, the vanadyl probe must coordinate in this manner in each of these regions. The resulting The Journal of Physlcal Chemlstry, Vol. SO, No. 17, 1976
Robert F. Campbell and Melvin W. Hanna complex most probably has square pyramidal geometry since vanadyl generally forms such complexes.39The actual composition of this complex is obscure; however, it is clear that more than one carboxyl group would be involved in the formation of a square pyramidal complex. D. Order in the Smectic Phase D. Generally,when one finds that a liquid crystalline phase can be ordered, it is interesting to determine the degree of order of the system. This can lead to a better understanding of the liquid crystalline structure and to insight into the nature of the intermolecular interactions responsible for the ordering. Conventionally, order in liquid crystalline systems has been quantitatively analyzed in terms of an effective time-independent Hamiltonian.9J0,40-43The paramagnetic probe is considered to undergo a complicated but very anisotropic motion approximated by resolving its motion into two parts: (1)motion about some molecular axis v, the director axis (i.e., the direction in which ordering occurs) and (2) motion perpendicular to the director. If motion about v is so fast that residual time-dependent effects are negligible and motion perpendicular to v is so slow that its effects on the spectrum are negligible, one obtains the following Hamiltonian for a paramagnetic probe having axially symmetric magnetic interactions % = goPeHzSz
+ AoIS + ’ h ( A g P a z
+ A A I z ) ( 3cosz$ - l)Sz (2)
where Ag = (gll = gL), AA = (All - A L ) , ( ) indicates an average, and 0 will be defined below. Description of the EPR spectrum under this Hamiltonian leads to determination of the “order parameter”, S’, given by
s’ = y2(3 cosz 0 - 1)
(3)
where 0 is the angle between v and the H direction. S’ is a measure of the mean motional amplitude, and therefore, alignment of the probe. With judicious choice of the probe, its alignment may be related to the order of the liquid crystalline solvent. The above approach is applicable when the motionally narrowing condition, l%l(t)l rc > l.30 Such is the case for our liquid crystalline region D, for here we also observe the effects of slow motion. Schwerdtfeger and Diehl (SD)ll have developed a method for determining the degree of order when the conventional approach breaks down. The method is based on the measurement of the relative intensities of the parallel and perpendicular components of the EPR spectrum. The approach assumes that the spectrum can be treated as due to an ordered distribution of rigid limit molecules. Thus, for a random distribution of rigid limit molecules, the number of radicles, dN, whose symmetry axis makes an angle between 0 and 0 do, assuming axial symmetric magnetic interactions, is
+
d N = NOsin 0 dB
(4)
where 0 is the angle between the symmetry axis and the director, v, of the liquid crystal and NOis the total number of radicals. In addition d N for the ordered distribution of rigid limit molecules is
V02+ Probe of Micelle-Liquid Crystal Systems
d N = NOf(0) sin 0 d0
1897
(5)
where f(0) is an appropriately chosen distribution function describing the ordered distribution. After Kivelson,45 the transition probability is assumed to be independent of 0. Therefore, the intensity of the absorption in a range of magnetic field, H and H dH,is directly proportional to dN1dH where
+
dNldH = (dN/dO)(dO/dH)
(6)
where dOldH is calculated from the rigid limit spin Hamiltonian and dNld0 is given by (4). Thus, one finds from (4) through (6) that where rl = rigid limit, o = ordered, and r = random. SD chose a distribution function similar to the one used by Meier and S a ~ p in e ~ studies ~ of dielectric relaxation in nematic liquid crystals, i.e. f(0) = C exp(-qllzT) sin20
When this function is inserted into (6) and the experimentally determined intensities for 0 = 0 and Jl12 are used to obtain the relative intensities of these components, then one obtains the value q1kT. The value of q may then be related to the degree of order.11 This approach, however, is only rigorously correct for an order distribution at the rigid limit; i.e., when there are no motional effects and one, therefore, has a static distribution of directors. Although S = 0.98 when calculated from SD’s data, and therefore an essentially rigid limit situation exists, our results for region D clearly show slow motional effects, i.e., S = 0.89. In such cases, the relative intensities of the parallel and perpendicular components vary from the rigid limit with 7c.29Thus, the relative intensities of the rigid limit parallel and perpendicular components are not the same as those of the corresponding slow motional components. Thus, this approach is, also, not applicable to our system. Further, the method suffers from an appropriate choice of f(0). An accurate determination of the degree of order in anisotropic liquids, where the motion is slow, must await an analysis after the method of PBF.29130
V. Conclusions It has been shown that the vanadyl probe provides a direct method for investigating the polar interfacial region of the molecular aggregates encountered in the sodium octanoatel octanoic acid/water, micelle-liquid crystal system. The method should be applicable to other anionic systems. The necessary condition is that the vanadyl ion form a stable complex, a t the pH of interest, with the constituents of the molecular aggregates to be investigated. Our results show that the vanadyl probe complexes at the carboxyl groups of the octanoate constituents making up the polar interfacial region in our system, and that it monitors the slow molecular motion at this surface. In all regions, except possibly region F, this motion appears to be on the same timescale. A qualitative estimate of this timescale gives 4 x s IT~ I1 X s; 7c being the correlation time characterizing this timescale. Given the variance in structure of the molecular aggregates investigated, and therefore possible differences in packing densities at the polar interface, one might expect differences in T~ from one region to another. For example, Ekwall12calculates the average area per polar head group, for a similar system, in region E to be 40-49 A2; in re-
x2.
ion B, 26-29 A2; in region D,24-25 A2;and in region F, 14-19 Unfortunately, a quantitative analysis of the slow motional spectra, which could point up such differences, must await the application of the methods of PBF.29p30 In addition, we have shown that the vanadyl probe is sensitive to the long ranged ordering occurring in, and the motion at the interface of, one of the lamellar liquid crystalline regions. Here, the vanadyl probe “sits down” at the polar interface with its V=O bond perpendicular to the bilayer surface. It is probable that the complex formed has square pyramidal geometry. The consistency of the rigid EPR parameters for all regions investigated indicates that in these other regions, as well, the probe complexes in the same way.
VI. Remarks It is interesting to speculate about the origin of the motion being monitored at the polar interface of the regions investigated. Probably the most important contributions to the dynamics of the interfacial region arise from rotations around the long molecular axes of the octanoate constituents and from isomerizations around their polymethylene bonds. However, the probable square pyramidal geometry of the complexed vanadyl probe requires that more than one carboxylate group be bound. Therefore, such motions are “locked out” by the structure of the complex and the only motion experienced by the probe would result from cooperative wagging modes of motion perpendicular to the octanoate molecular axis. Recently, a determination of the correlation time for motion perpendicular to the long molecular axis in a similar system,47 sodium octanoate/decanol/water,4s gave ~ = ~1.7( ,f0.2) l X s. This compares favorably with our estimate of T~ and adds confidence to the above interpretation. References and Notes (1) H. M. McConnell and B. G. McFarland, Q. Rev. Biophys., 3, 91, 1301 (1970). (a)J. Seelig, J. Am. Chem. Soc., 92, 3881 (1970); (b) J. Seelig, J. Limacher, and P. &der, ibid., 94, 6364 (1972); (c) H. Schindler and J. Seelig, J. Chem. Phys., 59, 1814 (1973); 61,2946 (1974); Ber. Bunsunges. Phys. Chem., 78, 941 (1974). (a) G. R. Luckhurst and A. Sanson, Mol. Phys., 24, 1297 (1972); (b) G. R. Luckhurst, M. Setaka, and C. Zannoni, ibid., 26,49 (1974); (c)G. R. Luckhurst and R. Poupko, Chem. Phys. Lett., 29, 191 (1974). W. E. Shutt, E. Gelerinter, G. C. Frygurg, and C. F. Sheley, J. Chem. Phys., 59, 143 (1973). A. S. Waggoner, 0. H. Griffith, and C. R. Chrlstensen, Roc. A’afl. Acad. Sci. US., 57, 1198 (1967). (a) N. D. Chasteen, R. J. DeKoch, B. Rogers, and M. W. Hanna, J. Am. Chem. Soc., 95, 1301 (1973); (b) R. J. DeKoch, D. J. West, J. C. Cannon, and N. D. Chasteen, Biochemistry, 13,4347 (1974); (c) N. D. Chasteen and J. J. Fitzgerald, bid., 13, 4338 (1974); (d) J. C. Cannon and N. D. Chasteen, “Proteins of Iron Storage and Transport in Biochemlstry and Medicine”, R. R. Crighton, Ed., North-Holland Publishing Co., Amsterdam, 1975. W. 2. Plachy, J. K. Lanyi, and M. Kates, Biochemisfry, 13, 4906 (1974). B. Lindman and P. Stllbs, J. Colloid lnterface Sci., 46, 177 (1974). J. P. Fackler, J. D. Levy, and J. A. Smith, J. Am. Chem. Soc., 94, 2436 (1972). G. C. Fryburg and E. Gelerinter, J. Chem. Phys., 52, 3378 (1970). C. F. Schwerdtfeger and P. Diehl, Mol. Phys., 17, 417 (1969). L. Mandell, K. Fontell, and P. Ekwall, Adv. Chem. Ser., No. 63, 89 (1967). Y. K. Levine, N. J. M. Birdsall, A. G. Lee, and J. C. Metcalf, Biochemistry, 11. 1416 11972) - -, D.Dodd;ell and A. Allerhand, J. Am. Chem. Soc., 93, 1558 (1971). J. J. Fitzgerald and N. D. Chasteen, Anal. Blochem., 60, 170 (1974). The vanadyl probe, to be added in a later step, is susceptible to 0 2 oxidation above pH 2.0. Since the pH of all samples prepared exceeds this value, they must be deoxygenated in order to prevent degradation of the probe. (17) P. Ekwall. I. Danielsson, and P. Stenius. MPTlnt, Rev. Sci., Phvs. Chem., Ser. One, 7, 97 (1972). (18) N. D. Chasteen and M. W. Hanna, J. Phys. Chem., 78, 3951 (1972). (19) P. Ekwall and P. Holmberg, Acta Chem. Scand., 19, 455 (1965). (20) P. Ekwall and L. Mandell, Kolloid-Z., 233, 938 (1968). (21) P. Ekwall and P. Solyon, Kolloid-Z., 233, 945 (1968). (22) P. Ekwall, J. Colloidlnferface Sci., 29, 16 (1969). (23) The tie lines have been deleted for ease of reading. \
The Journal of Physical Chemistry, Vol. 80, No. 17, 1976
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Michael D. Sevilla
(24) The spectra of the vanadyl probe in the frozen solutions of all regions are virtually Identical. (25) Note that the gross dlfferences in the center of gravity of the room temperature spectra when compared with the frozen solution spectrum is due to the difference in cavity frequency from the room temperature and frozen solution experimental arrangements. (26) L. J. Boucher, E. C. Tynan, and T. F. Yen, “Spectral Properties of 0x0vanadium(lV) Complexes. IV Correlation of EPR Spectra with Ligand Type” in “Electron Spin Resonance of Metal Complexes”, T. F. Yed, Ed., Plenum Press, New York, N.Y., 1969, pp 111-130. (27) There is always some reason for concern when low temperature data are used to infer a knowledge of the room temperature situation. In our case, there is the possibility of a temperature induced equilibrium shift from an unbound probe at room temperature to a vanadyl carboxylate (bound probe) complex when the solution is frozen at 77 K. However, comparison of room temperature, isotropic EPR parameters with those computed from the frozen solution, rigid limit parameters are generally In good agreement. See, for example, the tabular data In H. A. Kaska and M. T. Rogers, “Electron Spin Resonance of Flrst Row Transition Metal Complex Ions” in “Radical Ions”, H. A. Kaska and M. T. Robers, Ed., Wiley, New York, N.Y., 1968, Chapter 13, p 598. (28) Ekwall has shown [L. Mandell, K. Fontell, and P. Ekwall, Adv. Chem. Ser., No. 63,89 (1967)] in a similar system, sodium octanoate/decanol/water, that the thickness of the H20 layer ranges from 7 A at the lowest water content to 80 A at the highest water content in the smectic phase D. (29) J. H. Freed, G. V. Bruno, and C. F. Polnaszek, J. fhys. Chem., 75,3385 (197 1). (30) C. F. Polnaszek, G. V. Bruno, and J. H. Freed, J. Chem. fhys., 58, 3185 (1973). (31) S. A. Goldman, G. V. Bruno, and J. H. Freed, J. Phys. Chem., 76, 1858 (1972). (32) S. A. Goldman, G. V. Bruno, C. F. Polnaszek, and J. H. Freed, J. Chem. fhys., 56, 716 (1972).
(33) At low pH, vanadyl exists as the aquated species: VO(H20)5*+. (34) B. Svens and 8. Rosenholm, J. Colloid lnterface Sci., 44, 495 (1973). (35) Admittedly, the Stoke’s law relation treats the tumbllng molecule as a hard sphere, and therefore neglects the effects of iondipole Interactions between the Ionic micelle surface and the solvent, water. These interactions would tend to somewhat increase the value of T~ from this hard-sphere value. However, the effect would be expected to be less than an order of magnitude. (36) The spectra from all other regions were Independent of flat cell orientation. (37) Data on a simllar system, sodlum octanoate/decanol/water L. Mandell, K. Fontell, and P. Ekwall, Adv. Chem. Ser., No. 63, 89 (1967/l, gives the probable reason why region B does not show similar behavlor. This region has a high water content, 72-82%, and therefore the shearing forces are not large enough to produce orientation. (38) Due to the gel-like character of this phase, it was Impossible to prepare a bubble free sample. (39) N. D. Chasteen, R. L. Belford, and I. C. Paul, lnorg. Chem., 8, 406 11969) (40) S:H.-Glarum and J. H. Marshall, J. Chem. fhys., 44, 2884 (1966); 46, 55 (1967). (41) G. Havach, P. Ferrutl, D. Gill, and M. P. Klein, J. Am. Chem. SOC., 91,5726 (1969). (42) P. Ferruti, D. Gill, M. A. Harpold, and M. P. Klein, J. Chem. fhys., 50, 4545 (1969). (43) D. H. Chen and G. R. Luckhurst, Mol. fhys., 16, 91 (1969); Trans. Faraday SOC.. 65. 656 (19691. (44) C. F: Pohaszek and‘J. H. Freed, unpublished results. (45) D. Kivelson, J. Chem. fhys., 35, 156 (1961). (46) G. Meier and A. Saupe, Mol. Cryst., 1,515 (1966). (47) H. Schindler and J. Seelig, J. Chem. fhys., 59, 1841 (1973). (48) The pH is too high for a stable vanadyl carboxylate complex to form in this system.
Electron Spin Resonance Study of Nj-Substituted Thymine T-Cation Radicals‘ Michael D. Sevilla Department of Chemistry, Oakland University, Rochester, Michigan 48063 (Received March 29, 1976) Publication costs assisted by the U.S. Energy Research and Development Administration
a-cation radicals produced by photoionization of thymidine 5‘-monophosphate, thymidine, l-methylthymine, 1-ethylthymine, and thymine in basic 8 M NaC104-DzO glasses at 77 K were investigated by ESR spectroscopy. Analysis of the spectrum of each N1-substituted thymine shows coupling to the 5-methyl group (21 G), to the nitrogen at position 1(All = 13 G, A 1 < 4 G withg. - gll = 0.002), and to (3 protons on substituents at N1. Analyses were confirmed by computer simulations. The “experimental” spin density distribution was calculated and compared to McLachlan MO predictions. Parameters of the interaction of the 1position (3 protons were determined ( p a = 0.21, B2 = 79) and used to evaluate the conformations of the N1 substituent groups. Results found here are compared to previous results in aqueous glasses and single crystals.
Introduction
r-cation radicals2 of DNA bases have been identified in y-irradiated crystalline thymine: t h ~ m i d i n ecytosine: ,~ as well as y-irradiated DNA.6 It is our purpose to aid in the characterization of these radicals and to understand their chemistry by producing them individually in aqueous glasses. In our previous studies the a-cation radicals of several DNA bases were identified and their further reactions ~tudied.~-lO The present work extends the investigation to several N1substituted thymine derivatives (see structure I) and the nucleotide, thymidine 5’-monophosphate (TMP). The Journal of Physical Chemistry, Voi. 80,No. 17, 1976
H R
I. Structure of
thymines in basic
solution
Section 1-Methylthymine and 1-ethylthymine were obtained from
