J. Phys. Chem. 1982, 86, 3198-3205
3198
Mlcroenvlronmental Effects on Energies of Visible Bands of Nitroxides in Electrolyte Solutions and When Solubilized in Micelles of Different Charge Types. Significance of Effective Polarlty Estimates. Implications for Spectroscopic Probe Studies in Lipid Assemblies C. Ramachandran,$ Richard A. M e r , + and Pasupatl Mukerjee' School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53706 (Received: January 5, 1982; I n Final Form: April 12, 1982)
The absorption spectra of nitroxides in the visible region are highly solvatochromic and provide a good approach for assessing microenvironmental effects on the transition energies of nitroxides solubilized in lipid assemblies such as micelles. In this paper we present some experimental results on nitroxides in micellar solutions and indicate some methods of interpretation of such results which should be generally applicable to spectroscopic probe studies. In a previous paper solvent effects on the band energy, ET, of 2,2,6,6-tetramethylpiperidinyl-l-oxy (TEMPO) and 4-oxo-2,2,6,6-tetramethylpiperidinyl-l-oxy (OTEMPO) have been reported with special attention given to a series of reference solvents of graded hydrophilicity, dodecane, primary alcohols, methanol-water and ethanol-water mixtures, and water, suitable for investigating interfacial microenvironments. We report some data which indicate that the addition of inorganic electrolytes increases the ET value of TEMPO in water. This increase in apparent polarity is attributed to a cation effect causing increased hydrogen bonding through the electrostaticinfluence of cations on the surroundingwater molecules. It is also shown that a spectral parameter based on the ratio of absorbances at two fixed wavelengths can be used to assess the average polarities of the microenvironments of nitroxides solubilized in micelles but appropriate corrections must be made for micellewater distributions of the nitroxides. Based on the dielectricconstants of the reference solventsas medium polerity parmeters, the microenvironmental polarity in the solubilized state is expressed as an "effective" dielectric constant, Dew The corresponding differences in ET from aqueous solutions to the solubilized state (A&) were in the range of 2-3 kcal/mol. The D,ff and A& values of TEMPO indicate that its microenvironments in the micelle are moderately polar. The results are consistent with a predominant location of TEMPO at the micelle-water interface as inferred from previous work on similar systems and independent evidence presented in an accompanying paper. The Deffvalues of interfacially located TEMPO are roughly midway between the values of water and dodecane in all of the micellar systems. Electrostatic image interactions of the nitroxide dipoles at the interface caused by the proximity of the hydrocarbon cores provide a qualitative explanation of this general finding. This explanation is not satisfactory, however, on a quantitative basis. Further analysis indicates that the image interactions on the hydrogen-bonded nitroxide complex may be important and that the net effect of hydrogen-bonding interactions may be significantly weaker at interfaces when compared to bulk water. The Deffand AET values in different micellar systems of different charge types vary over a small range. These variations are consistent with the effects expected from field strengths on the order of 2 x lo6 V/cm in the interface region of ionic micelles with some additional effects due to cations at the surface of anionic micelles. A micelle with zwitterionic head groups similarly shows the effect of the expected dipole field at the interface. In the case of the phospholipid palmitoyl lysolecithin the effect of the zwitterionic moiety appears to be small because of its distance from the hydrocarbon core. Solubilized OTEMPO and two long-chain stearic acid derivatives containing a nitroxide moiety at different positions along the alkyl chain indicate DeRvalues similar to those of TEMPO in two micellar systems. A quaternary ammonium nitroxide derivative, however, shows a significantlylower Deffvalue in anionic micelles. These data have been interpreted in terms of interfacial locations of the nitroxides involving different orientations in some cases. The conclusions of this paper and some conclusions of the accompanying papers underscore the hazards of the use of nitroxides and similar species as spectroscopic "probes" of the hydrocarbon interiors of micelles and other lipid assemblies. Many common simplifying assumptions about the behavior of such spectroscopic probes in lipid assemblies can be grossly misleading. The implications of some of the results obtained for spectroscopic probe studies are pointed out.
Introduction
This investigation is primarily concerned with the nature of the microenvironments experienced by nitroxides solubilized in micellar systems, the effects of such microenvironments on their transition energies, and the effective polarities sensed by the solubilized nitroxides. We also report some effects of added electrolytes on the visible spectra of nitroxides in aqueous solutions. The literature on the use of spectroscopic probes for studying lipid assemblies is voluminous. The present paper deals with a number of points which require attention before the results
* Lubrizol Corporation, Wickliffe, OH
44092. Abbott Laboratories, North Chicago, IL 60064. 0022-3654/82/2086-3198$01.25/0
of such investigations can be interpreted properly. It also develops several approaches toward the assessment of microenvironmental characteristics of interfaces including effects of the dielectric asymmetry associated with interfaces, hydrogen-bonding interactions at interfaces, and the presence of electric fields. Nitroxides have been used extensively as spin probes in studies of membranes and membranelike structure^^-^ (1) Presented in part at the 182nd National Meeting of the Americal Chemical Society, New York, 1981, and based in part on the Ph.D. dissertation of R. A. Pyter, School of Pharmacy, University of Wisconsin, Madison, WI, 1980. (2) Mukerjee, P.; Ramachandran, C.; Pyter, R. A. J. Phys. Chem., preceding paper in this issue.
0 1982 American Chemical Society
Microenvironmental Effects on Nltroxide Band Energies
A
xH3’3
n ANI A
A N I&
0 OTEMPO
The Journal of phvsical Chemistry, Vol. 86, No. 16, 1982 3199
I
0
0 TEMPA+
TEMPO
0’ ‘N,
I
I
w 16 NS
O 5NS
Flgure 1. Structures of nitroxides used in solubilization studies.
using electron spin resonance (ESR). The primary interest in these studies has been the characterization of the fluidity of membranes. Several studies have also been reported on micellar systemsg1’ and other lipid assemblies12-14employing ESR. In some of these investigations the problem of the location of the nitroxide group in the lipid assemblies has been addressed. Very little use has been made in such investigations of the visible spectra of nitroxides. In the previous paper we have presented extensive data in many solvents to demonstrate the solvatochromic character of the weak n + +transitions involved.2 Particular attention was given to a series of reference solvents of graded polarity, dodecane, primary alkanols, methanol-water and ethanol-water mixtures, and water. The usefulness of such solvents as calibrating solvents for microenvironmental studies in lipid assemblies has been discussed.15J6 The visible band system of the nitroxides is well suited for the assessment of microenvironmental polarities of nitroxide compounds in lipid assemblies. On the basis of similar investigations of aromatic solutes in micellar systems,15J6such polarity estimates are expected to provide an important approach to the determination of the location of the nitroxide moiety in lipid assemblies. The visible spectra can also be used to obtain micellewater distribution coefficients of nitroxide compounds and the solubilizing powers of micelles in a straightforward manner? Such data, along with some solvent partition and interfacial tension data, presented in the following publ i ~ a t i o n ,provide ~ the basis for the examination of the solubilization characteristics of nitroxides in lipid assemblies using a general two-state model of solubilization presented recent1y.l6J7 It should be emphasized that because of the low molar absorbances, of the order of 10 (3)Pyter, R. A.; Ramachandran, C.; Mukerjee, P. J. Phys. Chem., following paper in this issue. (4)McConnell, H.M.; McFarland, B. G. Q. Reu. Biophys. 1970,3,91. (5) Hubbell. W.L.: McConnell. H. M. Proc. Natl. Acad. Sci. U.S.A. 1969;61.16. (6)Haia, J. C.; Schneider, H.; Smith, I. C. P. Biochim. Biophys. Acta 1970,202,399. (7)Jost, P.; Libertini, L. J.;Herbert, V. C.; Griffith, 0. H. J.Mol. Biol. 1971,59. 77. ( 8 ) Fox, K. K. Trans. Faraday SOC.1971,67,2802. (9)Waggoner, A. S.;Keith, A. D.; Griffith, 0. H. J.Phys. Chem. 1968, 72,4129. (10)Oakes, J. Nature (London) 1971,231,38. (11)Atherton, N.M.; Strach, S. J. J. Chem. Soc., Faraday Trans. 2 1972,68,4374. (12)Keith, A. D.; Snipes, W.; Chapman, D. Biochemistry 1977,16,634. (13)Luna, E. J.; McConnell, H. M. Biochim. Biophys. Acta 1977,470, 303. (14)Seelig, J. J. Am. Chem. SOC.1970,92,3881. (15)Cardinal, J. R.; Mukerjee, P. J. Phys. Chem. 1978,82,1613. (16)Mukerjee, P.; Cardinal, J. R. J . Phys. Chem. 1978,82,1620. (17)Mukerjee, P. In “Solution Chemistry of Surfactants”; Mittal, K. L., Ed.; Plenum Press: New York, 1979;Vol. 1, p 153.
L/(mol cm), it is necessary in such studies to employ nitroxide concentrations much higher than those typically used in ESR investigations. Figure 1 shows the structures of the nitroxides investigated. Several different nitroxides were chosen to study in a comparative fashion effects of the presence of additional polar moieties and the effects of nitroxide substitution in long-chain acids far from the polar head groups.
Experimental Section Materials. 4-0xo-2,2,6,6-tetramethylpiperidinyl-l-oxy (OTEMPO) and 2,2,6,6-tetramethylpiperidinyl-l-oxy (TEMPO) used have been described.2 4-(Trimethylamino)-2,2,6,6-tetramethylpiperidinyl-l-oxy iodide (TEMPA+) was purchased from Molecular Probes Inc., Texas, and used as such. The two stearic acid probes, 2-(3carboxypropyl)-4,4-dimethyl-2-tridecyl-&oxazolidinyloxyl (5NS) and 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl(16NS) were purchased from Syva Co., California, and used without further purification. Sodium dodecyl sulfate (SDS) obtained from BDH was purified by extraction with ether followed by recrystallization from water, the procedure being repeated several times. The product was dried over P2O5 for several days. Its purity was tested by surface tension measurements. No minimum was observed near the critical micellization concentration (cmc). Magnesium dodecyl sulfate (Mg(DS),) was precipitated from aqueous solutions of sodium dodecyl sulfate by using saturated magnesium chloride, the procedure being repeated 3 times. The precipitate was washed repeatedly with cold water and recrystallized from hot water. It was then dried over P205for several days. The cationic surfactant cetyltrimethylammonium chloride (CTAC) was purified by using a published method18involving three recrystallizations from ethyl acetate-ethanol mixtures. The nonionic surfactant, octyl p-D-glucopyranoside (OG) was purchased from Calbiochem-Behring Corp., California, and used as such. Surface tension measurements of OG solutions exhibited no minimum near the cmc region. OG was stored over P205under vacuum to prevent degradation through water uptake and subsequent hydrolysis. The zwitterionic surfactant, N-dodecyl-N,N-dimethyl-3-ammonio1-propanesulfonate (ZWIG 12) containing the positive end of the dipole close to the chain, was obtained from Calbiochem-Behring Corp. and used as such. The other zwitterionic surfactant system, y-palmitoyl L-a-lysolecithin (P-LYSO),containing the negative charge center closer to the hydrocarbon chain, was also purchased from Calbiochem-Behring Corp. and used without further purification. Hydrophobic impurities are known to perturb monomer-micelle equilibria seriously in the cmc region.lg In all of our experiments the concentrations of the surfactant systems were kept well above the cmc, and, therefore, the effects of any impurities were substantially reduced because of dilution in the micelles. All other chemicals and solvents were purified, whenever necessary, by using methods adapted from the literature.20 Apparatus. Spectra were obtained on a Cary-14 or a Cary-118 recording spectrophotometer. A Cary-16 manual spectrophotometer was also used for absorbance measurements at fixed wavelengths. The wavelength accuracy was checked by using a standard holmium oxide filter.21 (18)Ralston, A. W.; Eggenberger, D. N.; Harwood, H. J.; Dubrow, P. L. J. Am. Chem. SOC.1947,69,2095. (19)Mukerjee, P.; Mysels, K. J. Natl. Stand. Ref. Data Ser. (US., Natl. Bur. Stand.) 1971,No. 36. (20)Desai, N. R. Ph.D. Thesis, University of Wisconsin, Madison, WI, 1981. (21)Vandenbelt, J. M. J. Opt. SOC.Am. 1961,51, 802,915.
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The Journal of Physical Chemistry, Vol. 86, No. 16, 1982
60.60-
0.6 -
I
I
I
Ethanol
I
I_
Dodecane
60.40-
I
400
67201 0
I I I I I I 1.0 2.0 3.0 4.0 5.0 6.0
I
20
I
0.0
I
MOLALITY ( m )
Figure 2. Plots of energy of transition, ET(kcal/mol),of TEMPO as a function of molality ( m ) of several electrolytes in aqueous solutions: (a) KCI, (b) NaCI, (c)LiCI, (d) NaBr, (e) NaI, (f) LaCI,, and (9) MgCI,. Values of slope, dE,ldm, are (a)0.053, (b) 0.109, (c)0.115, (d) 0.122, (e) 0.127, (f) 0.297, and (9) 0.302.
The spectrophotometers were equipped with constanttemperature circulation units to regulate the cell-compartment temperature. All spectra were obtained at 25 "C. The cells had path lengths of 1cm and were used with Teflon caps to minimize any evaporation losses. The reference cell always contained a solution identical in composition with that in the sample cell except for the added nitroxide. This procedure compensated for small turbidity effects. All measurements were carried out with freshly prepared solutions. Methods. The method used to determine the A- values for the relatively flat absorption bands of the nitroxides have been described.*
Results and Discussion Effect of Electrolytes in Aqueous Media. This investigation had a dual purpose. It is well-known that the local concentration of counterions a t charged interfaces, for example those of micelles, can be very high, on the order of several mol/L.2za It is of interest to know whether the spectra of nitroxide groups at these interfaces are affected by the local ionic composition. A second point of interest arises from the nature of the interactions involved. An extensive body of data on the effects of added electrolytes on the spectrum of aqueous mesityl oxide suggested several years ago that cations in solution increase the hydrogenbonding ability of water.24 Although this work remains unpublished, NMFt investigations on micellar systems have provided some confirmatoryevidence.% More recent work on ANvalues of nitroxides, the nitrogen hyperfine splitting constants in ESR experiments, are also consistent with the general picture.26 Figure 2 shows the ET values of TEMPO (energy of transition correspondingto, , X in kcal/mol) in aqueous solutions of several electrolytes. ET increases linearly with the molal concentration of all of the electrolytes studied. Comparison of the effects of NaC1, NaBr, and NaI indicate (22) Mukerjee, P.; Cardinal, J. R.; Desai, N. R. In "Micellization, Solubilization and Microemulsions"; Mittal, K. L., Ed.; Plenum Press: New York, 1977; Vol. 1, p 241. (23) Mukerjee, P. J.Phys. Chem. 1962, 66, 943. (24) Mukerjee, P.; Cardinal, J. R., unpublished work. Cardinal, J. R. Ph.D. Thesis, University of Wisconsin, Madison, WI, 1973. (25) Gustavsson, H.; Lindman, B. J. Am. Chem. SOC.1975,97,3923. (26) Jackson, S. E.; Smith, E. A.; Symons, M. C. R. Faraday Discuss. Chem. SOC.1977, No. 64, 173.
425
450
475
500
Wavelength (nm)
Flgure 3. Visible spectra of TEMPO h water, ethanol, dodecane, and 0.30 M sodium dodecyl sulfate (SDS).
that the anion has a relatively small influence. The increase in ET is, therefore, primarily due to a cation effect and is in the direction of an increase in polarity.2 This is consistent with the idea of an enhancement in hydrogenbonding interactions of water with the nitroxides by cations. Presumably the cations increase the ability of the water molecules in the immediate hydration envelopes surrounding the cations to undergo hydrogen-bonding interactions because of electrostatic effects. In the case of mesityl oxide, the effect was observed to increase in the sequence Cs+ < Na+ < Li+, consistent with increasing charge density of the cations." For the nitroxides, Li+ and Na+ have about the same effect but both have significantly greater effects on ET than K+. Also in agreement with previous work on the mesityl oxide system, the bivalent Mg2+and the trivalent La3+are much more potent than Na+, as would be expected from higher charge densities. Microenvironmental Effects on Effective Polarities of TEMPO Solubilized in Micelles. Figure 3 shows the spectra of TEMPO in dodecane, ethanol, water, and in 0.3 M aqueous SDS. In 0.3 M SDS about 90% of TEMPO is solubilized, as estimated from micelle-water distribution data to be reported later.3 The spectrum of TEMPO in 0.3 M SDS (Figure 3) shows clearly that the average polarity of the microenvironment of solubilized TEMPO is high in this medium, intermediate between those of water and ethanol. In most previous studies,"ll micelle-water distribution coefficients have not been estimated. Measurements at high SDS con~entrations~~~' have been interpreted directly to indicate a high microenvironmental polarity, in agreement with Figure 3. When nitroxides are added to surfactant solutions above the cmc, the nitroxide partitions between the micelles and the intermicellar fluid. Observed spectra, therefore, are composites of contributions from the fraction solubilized in micelles and the fraction in water. In order to assess a spectral parameter characteristic of the solubilized state itself, it was convenient to use a ratio of absorbances at two suitably chosen fixed wavelengths, defined as R , as a polarity-sensitive parameter.15J6 R is independent of the concentration of the chromophore in any medium as long as the absorption band remains the same. Any shift in the band position, however, changes the value of R. It was found that the half-widths of the bands in the reference solvents are very similar in the frequency scale so that the bands are of nearly identical shape. For TEMPO, R values are calculated as ratios of absorbances at 425 and 475 nm. Figure 4 shows a plot of this ratio in the reference solvents against (27) Dodd, G. H.; Barrat, M. D.; Raper, L. FEBS Lett. 1970,8, 286.
The Journal of Physical Chemistry, Vol. 86, No. 16, 1982 3201
Microenvironmental Effects on Nltroxide Band Energies
t
1.601.50-
0
1.40 -
1.30
R 1.30-
1.20 1.10 -
0.70v
0.50;
I
I
I
1
I
I
I
30 40 50 60 70 IO 20 BULK DIELECTRIC CONSTANT, D
Flgure 4. Plot of the ratlo
I
I
80
t425/t4,s, R,
for TEMPO as a function of solvent dielectric constants. Solvents from left to right: dodecane, carbon tetrachloride, 2-methyl-2-propano1, lhexanol, 1-pentanol, 1propanol, ethanol, methanol, 80% ethanol, 60% ethanol, 70% methanol, 50% ethanol, 40% ethanol, 50% methanol, 40% methanol, 30% methanol, 20% ethanol, 10% ethanol, 10% methanol, and water. Ail alcohol-water mixtures are by weight percent.
the dielectric constant of the solvents, D. Absorbance ratios at 408 and 460 nm were used for OTEMPO. The curve obtained for OTEMPO was very similar to that of Figure 4. In both cases R values were also monotonic functions of, , A and ET. For micellar studies only the region between methanol (D = 32.6) and water ( D = 78.5) was significant. In this region R varies by nearly a factor of 2 and is a roughly linear function of D. The advantages of using R values are that the values can be determined more precisely than, A values in solutions of surfactants and can also be analyzed in a simple manner in terms of the micelle-water distribution of the nitroxide to determine R,, the value of R for the solubilized state, as also K,, the micelle-water distribution ~oefficient.~ To determine, , A or E T of the completely solubilized state from direct measurements is more difficult. If Rm is used, however, E T for the solubilized state can be determined from the monotonic calibration plot of R with E T in the reference solvents. The working equation for determining R, derived in the accompanying paper3 is
R = R,
%a 1 +-(R, ~ 2 , mKm
4a
- R)-
4m
R is the ratio e l / t 2 where tl is the average molar absorbance at AI and t2 at A2, measured in micellar solutions of varying surfactant concentrations and a constant nitroxide concentration. t2,aand t2,, are the molar absorbances at A2 in water and in the micellar (solubilized) state, respectively. R, is the experimentally determined ratio in water, 4, and 4, are the volume fractions of water and the micelles. A plot of R against (R, - R)4,/4, can be extrapolated to zero value of the latter quantity to determine R,. This value of R,, when compared with the calibration curve such as that shown in Figure 4, gives an estimate of the average effective dielectric constant of the solubilized species, D& It should be stressed that D& thus obtained is an empirical measure of polarity. It can be readily converted to the H scale discussed before2 or any other empirical scale of polarity. R, can also be used to determine ET in the solubilized state because R values were found to be a monotonic function of E T in the reference solvents. Since E T of hydroxylic solvents has been shown to be a linear function of D,2 we have preferred to use Defffor micelles
I
I .oo 0
I
5
I
I
I
IO 15 20 (Ra R ) &
-
I
25
I
30
I
35
I
40
- R ) 4 ,/$ for TEMPO solubilized in micellar systems: (0)cetyltrlmethylammonium chloride (CTAC), (0) sodium dodecyl sulfate (SDS). Flgure 5. Plots of R against (R,
to determine E T for the solubilized state from this correlation reportedS2 The above approach illustrates the superiority of the use of visible spectra of nitroxides as compared to the use of nitrogen hyperfine splitting constants (AN) in electron spin resonance for studying lipid assemblies. It is extremely difficult to obtain characteristic parameters for the completely solubilized state or micelle-water partition coefficients by measuring AN data for a nitroxide in distribution equilibrium between micelles and water.l0 Figure 5 shows two characteristic plots of R against (R, - R)$,/$, for TEMPO in SDS and CTAC. In these experiments, efforts were made to keep the nitroxide concentrations as low as feasible considering the low molar absorbances. The nitroxide concentrations were typically 3-10 mM. The highest surfactant concentrations used varied in the range 60-300 mM. When fractions of the nitroxides solubilized were estimated, the mole fractions of the nitroxides in the micelles at the highest concentrations were in the range of 0.01-0.04 in most cases. A t these low values the micelle-water partition coefficients are expected to be relatively independent of nitroxide concentration.28 The plots of eq 1in all cases were linear within experimental error, indicating the constancy of K,. In several cases, the nitroxide concentration was varied over a wide range. The value of R, was independent of this variation. Figure 5 shows that there are significant differences between the extrapolated R, values and the lowest experimental R values obtained for SDS and CTAC at the highest concentrations employed, 0.28 and 0.20 M, respectively. Thus, direct interpretation of measurements even at such high micellar concentrations do not provide data characteristic of the solubilized state itself, and our experimental procedure in determining R, by extrapolation is necessary. The Deffvalues estimated for TEMPO solubilized in several micellar systems of different charge types are given in Table I. The uncertainties vary somewhat from system to system. The estimated standard errors were about f l in Defffor SDS, Mg(DS)2, CTAC, and P-LYSO, the remaining systems having a slightly higher error of f2 mits. The data indicate that the microenvironment of TEMPO solubilized in all of the micelles is quite polar, the polarity (28) Mukerjee, P.J . Pharm. Sci. 1971, 60,1531.
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TABLE I: Effective Microenvironmental Polarities of TEMPO Solubilized in Micellar Systems (D,ff)and A ET Values micellar svstem octyl glucoside (OG) sodium dodecyl sulfate (SDS) magnesium dodecyl sulfate (Mg(DS),) cetyltrimethylammonium chloride (CTAC) Zwittergent 3-12 (ZWIG12) palmitoyl lysolecithin (P-LYSO)
Dntr kcal/mol 40 48 46 33 32 39
2.55 2.03 2.16 3.00 3.07 2.61
a A E T is obtained by subtracting the values of ET characteristic of the solubilized state, as estimated from the Deff value, from the value of ET in water, 67.35 kcal/ The relation used for estimating micellar ET from Deff is ET = 62.197 t 0.06510, as reported for hydroxylic solvents.’
being comparableto that of methanol or higher. The range in observed Deffvalues indicates also that the particular interfacial microenvironment of the micellar system, as, for example, its charge type (cf. Figure 5), also plays a role in determining the value of Deff. Table I gives also the estimated lowering of E T in micellar systems, A E T , as compared to water. The average value of about 2.5 kcal/mol and the range of variation of about 1 kcal/mol indicate the importance of several microenvironmental features. The generally high values of Deffare incompatible with a location of the nitroxides in the core of the micelles. In terms of the recently proposed two-state model of solubilization which invokes an equilibrium between a ‘dissolved state”, associated with the micellar core, and an ”adsorbed state”, associated with the micelle-water interface, it is clear that the fraction of the solubilized species in the adsorbed state makes a very important contribution to the spectra. In order to ascertain how important this fraction is, it should be noted that the D,H value itself, and the corresponding fractional hydrophobic or hydrophilic index that can be assigned to it, should not be converted to the fraction at the interface as is sometimes done. In fact it is likely that close to 100% of the nitroxide is at the interface. The reason for this is that a nitroxide group situated at the interface in an adsorbed state is in an asymmetric environment quite different from that of water. Thus, for example, it has been shown that the interionic charge-transfer interactions of dodecylpyridinium iodide (DPI) in its own micelles correspond to an “effective” dielectric constant of about 36, following a solvent calibration procedure similar to the one used here.29,30This interionic charge-transfer system involves short-range interactions between the charged moiety of the pyridinium ion and the iodide counterion. Their exclusive location at the micelle-water interface region, the innermost part of the electrical double layer, where both the charges are exposed to water, can be assumed with some c o n f i d e n ~ e . ~The ~ * DPI ~ ~ polarity probe thus serves as a model probe for interfacial microenvironments. The similarity of this Deffvalue of 36 of the DPI polarity probe in its own micelles with that of TEMPO in the cationic system CTAC (Dd = 33) argues strongly for a predominant location of TEMPO in the interface region with the nitroxide group in contact with water. This similarity also extends to other micellar systems and will be discussed in a later publication. As an additional example we cite the case of the nonionic octyl glucoside system. The DPI probe gives a DeEvalue of 44 in this case,3l slightly different from (29) Ray, A.; Mukerjee, P. J.Phys. Chem. 1966,70,2138. (30)Mukerjee, P.; Ray, A. J.Phys. Chem. 1966,70, 2144.
Ramachandran et al.
a preliminary value of 46 reported as compared to the D,ff value for TEMPO, 40 (Table I). Additional evidence of a predominant location of the nitroxide group in the micelle-water interface region comes from the comparison of some micelle-water distribution coefficients with dodecane-water distribution coefficients of nitroxides, and the assessment of the interfacial activities of TEMPO and OTEMPO at the dodecane-water interface, as described in the following paper.3 These investigations indicate that the fraction of solubilized TEMPO in the adsorbed state is likely to exceed 98%. In view of this corroborative evidence and arguments, we will ignore the small fraction of TEMPO that exists in the dissolved state, Le., in the hydrocarbon core, in the remainder of this discussion. The molecular interaction most responsible for the location of TEMPO in the interfacial region is likely to be the strong hydrogen bonding of the nitroxide group with water for which an energy of about -7.5 kcal/mol has been e ~ t i m a t e d . ~
Factors Affecting Transition Energies and Microenvironmental Polarities. Several microenvironmentalfactors that might affect apparent polarities at micelle-water and similar interfaces have been d e s ~ r i b e d . ~The ~ ~range ~~*~~ of variation in the AET and Deffvalues of TEMPO (Table I) in different micellar systems of very different head-group structures is significant but small with respect to the magnitudes of AET and the difference of the Deffvalues from the dielectric constant of water (78.5). These data, therefore, argue strongly for an important contribution of the general effect of the proximity of the low dielectric cavity,the hydrocarbon core of the micelle. This proximity effectn* is likely to be significant for all interfacial regions which separate hydrocarbon-like regions and an aqueous medium, such as those of monolayers, bilayers, and membranes. Our analysis indicates that at least two types of interactions, perhaps not independent, are likely to be involved. It is known that, when a charged group or ion is transferred from an aqueous medium to an interface region close to a fluid of a low dielectric constant, there are repulsive interactions which become stronger as the charged group approaches the i n t e r f a ~ e . ~These ~ - ~ ~interactions of electrostatic origin are described as image interactions at planar interfaces and are responsible for the elevation of surface and interfacial tension^.^^.^ Similar interactions are also experienced by dipolar g r o ~ p s . ~ ~ , ~ ~ The zwitterionic amino acid glycine, for example, increases the surface tension of water. An equivalent description of these repulsive interactions as a dipole approaches a dielectric interface can be given by postulating that the dipole is transferred from water to a “homogeneous”medium of a lower dielectric constant. This approach provides a ready qualitative explanation as to why the “effective polarity” experienced by an ion or a dipole is lower near such interfaces in terms of the increase in the self-potential of the species caused by image interactions. For a more quantitative treatment, we have used expressions derived by Buff et a1.36,37for calculating the change in interaction energy upon bringing a spherical cavity of radius a with a point dipole of moment p from an aqueous medium of dielectric constant D, to a planar interface separating a medium of negligibly low dielectric (31) Mukerjee, P.; Desai, N. R.; Ramachandran, C., unpublished work. (32) Mukerjee, P.; Desai, N. R. Nature (London) 1969,223, 5210. (33)Harned, H. S.;Owen, B. B. “The Physical Chemistry of Electrolytic Solutions”; Reinhold: New York, 1958. (34)Onsager, L.;Samaras, N. N. T. J . Chem. Phys. 1934,2, 528. (35)Stigter, D. J.Phys. Chem. 1964,68,3603. (36)Buff, F. P.; Goel, N. S. J. Chem. Phys. 1972,56,2405. (37)Buff, F.P.; Goel, N. S.; Clay, J. R. J.Chem. Phys. 1975,63,1367.
The Journal of Physical Chemistry, Vol. 86, No. 76, 1982 3203
Microenvironmental Effects on Nitroxide Band Energies
constant from water. Buff et a1.36137have shown that for the specific geometry of the spherical cavity in contact with the interface, i.e., when its distance from the interface, z, equals its radius a, the interaction energy, Wi, for a dipole oriented perpendicular to the interface can be expressed in a simple form as
Wi = 0.4546p2/D43
(2)
Wi can now be equated to the energy of transfer of the same spherical dipole from water to a homogeneous medium of lower dielectric constant Di. The energy, w, of such a dipole, assumed to be nonpolarizable, in a medium of dielectric constant D is given by38*39 the equation (3) The required value of Di can be calculated by equating Wi with the change in W when D, changes to Di.
-1
D,-1 Di+1 0.4546 = Dw[ 2 D , + l - 2Di+1
(4)
For spherical species, Di is thus independent of the choice of a or p and has the value of 48.6. Effects of interfacial curvature cannot be estimated ~ e 1 1 ~ but 9 ~ 'are likely to be small for the systems of interest here, as revealed in some parallel studies to be reported later.31s40 The qualitative agreement of the value of Di with the Deffvalue of TEMPO in nonionic micellar systems such as OG, in which the proximity of the core is likely to be the most important microenvironmental feature, is of considerable interest and suggests that image interactions may in part be the origin of the Deffvalue. However, the magnitude of Wi is small. In order to calculate the value of Wi, we note that eq 3 has been applied in the previous papel.2 to calculate the change in E T of TEMPO in aprotic solvents. The coefficient y of eq 5 of that paper2 thus represents an estimate of the absolute value of p 2 / a 3assuming the model of a nonpolarizablespherical cavity with a point dipole in the center is applicable. The value of y is 4.42 kcal/mol, so that Wi is calculated to be only about 0.03 kcal/mol in comparison to AET for OG of 2.55 kcal/mol. Although larger values of Wi could result if z is less than a, which would correspond to lower values of DeR,the discrepancy is much too large to be accounted for in this manner unless rather nonphysical values of z are invoked. Moreover, it has been shown in the previous paper2 that eq 3 or its modified forms for polarizable dipoles give a very low estimate of the solvent effect on the E T value of TEMPO in water and other hydroxylic solvents. The major part of this solvent effect arises from hydrogen-bonding interactions. Thus, eq 3 applied to the nitroxide dipole alone is of qualitative significance only. Changes in the hydrogen-bonding interactions in the interface region as compared to bulk water must play an important role in determining Deffor A E T values (Table I). Two modelistic approaches appear to be of some interest in accounting for the hydrogen-bonding contributions to AET or the Deftvalues (Table I). If the nitroxide at the interface is assumed to be hydrogen bonded, then the NO. .-HO hydrogen-bonded complex species may be considered as the solute species for which the work of transfer
Wi should be calculated (eq 3). Equation 3 indicates that, if this complex species is also described as a spherical dipole of radius a'and is in contact with the interface (i.e., z = a?, an identical Des value of 48.6 for a planar interface will again be obtained (eq 4). However, since the dipole moments of hydrogen-bonded complexes are high,41*42Wi values could be raised substantially. This very artificial model completely ignores polarizability effects and multiple interactions of 'the nitroxide dipole with solvent molecules. Yet, it is likely that the electrostatic image interactions of not only the solute dipoles but also the associated solvent dipoles may be important in determining AET or Deffvalues at interfaces. A related approach is derived from the theory of the dielectric constants of hydrogen-bonded liquids due to Kirkwood and F r o h l i ~ h . This ~ ~ approach ~ ~ ~ ~ ~has been examined with reference to solvent effects on E T in hydroxylic solvents.2 In dielectric theory, a correlation parameter g is invoked which is a measure of the local order. For the interactions of the nitroxides in hydroxylic solvents, the possible importance of the role of such a parameter gN has been discussed.2 In dielectric theory, g can be expressed approximately as g = 1 n cos 0 (5) where n is the number of nearest neighbors around a dipole, 0 is the angle between thedipole moments of a pair of neighboring molecules, and cos 0 is an average overall orientation of both molecules. Although the calculation of gN for the nitroxides appears to be a formidableproblem, an approximate effective value of about 4 was derived for water in the previous paper.2 Such hydrogen-bonding interactions of the nitroxide dipole at an interface can be weakened by a reduction in the effective value of gN arising from the reduced solid angle around a nitroxide dipole available to water molecules. This approach also predicts a curvature effect of the interface. To what extent such considerations should be modified by the detailed nature of the local structure of water at interfaces is not known. However, an overall qualitative result emerging from all of these considerations is that hydrogen-bonding and general solvating abilities of water may be reduced at an interface in roughly the same manner as in a change of the medium from water to methanol-water mixtures. It should be stressed that, for the thin regions being considered, the finite dimension of the water molecule itself is likely to be an important factor and continuum models of solvents may not be appropriate.22 In comparison to the OG system, D,ff values are higher for the anionic micelles SDS and Mg(DS)2and lower for the cationic CTAC. A number of factors may be responsible for these additional effects of the detailed nature of the interfacial microenvironment in charged systems. Intense electric fields exist at such charged interfaces which are expected to cause some lowering of the local dielectric constant through dielectric saturation e f f e ~ t s .This ~~~~~ effect should reduce Deff for both cationic and anionic systems. The experimental data indicate, however, that the overall effect is different for cationic and anionic micelles, and, therefore, the direction of the field plays an important role. Solubilized TEMPO in the interface region is likely to have the positive end of the dipole oriented toward the hydrocarbon core. The roughly parallel di-
+
(41) Malecki, J. Acta Phys. Pol. 1965, 28, 891. (38) Bijttzher, C. J. F. "Theory of Electric Polarization", 2nd ed.; revised by Van Belle, 0. C., Bondewijk, P., and Rip, A.; Elsevier: Amsterdam, 1973, Vol. 1. (39) Kirkwood, J. G. J . Chem. Phys. 1934,2, 351. (40) Ramachandran, C.; Mukerjee, P., unpublished work.
(42) Jadzyn, J.; Malecki, J. Acta Phys. Pol. A 1972, 41, 599. (43) Kirkwood, J. G. J. Chem. Phys. 1939, 7,911. (44) Frahlich, H. "Theory of Dielectrics"; Oxford University Press: London, 1949. (45) Booth, F. J. Chem. Phys. 1951, 19, 391, 1327, 1615.
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The Journal of Physical ChemWy, Vol. 86, No. 16, 1982
rection of the field in CTAC micelles is expected to increase the ground-state energy of TEMPO whereas the opposite directions of the fields in SDS and Mg(DS)2 micelles are expected to reduce its ground-state energy, both effects being in the right direction for explaining the differences in the A E T values. In addition, the effects of the high local concentrations of the cationic counterions of SDS and Mg(DS)2 at the interface, through the increased hydrogen-bonding effects noted earlier in this paper, could also be significant. In order to assess these interactions which arise from factors other than the proximity effects in a semiquantitative fashion, we note that the somewhat lower curvatures of the micelles of the longer-chain surfactants should increase the proximity effect slightly, as is found in parallel studies with DPI.31 Thus, the proximity-effect contribution to the AET value of CTAC may be somewhat higher than 2.55 kcal/mol, the value for octyl glucoside. A rough upper estimate of about 2.75 kcal/mol can be made for the proximity-effect contribution to AET for CTAC, on the basis of additional data in other system^.^^,^ Thus, the effect of the charges on the CTAC micelle is to increase AET by 0.35 f 0.1 kcal/mol. Since halide ions have very little effect on the ET value of TEMPO in water (Figure 2), this change in AET, A W , can be attributed to the external field in the innermost part of the electrical double layer of CTAC micelles. If the relation AW = -Y+NF (6) is used to calculate the strength of this external field F from the dipole moment, p ~of, TEMPO, 3.14 D units, the calculated absolute value of F is 2.3 X lo6 V/cm, which is reasonable for the interface of charged micelles.30 Similar calculations suggest that the A E T is reduced by 0.62 f 0.1 kcal/mol for the anionic SDS micelles and by 0.75 f 0.1 kcal/mol for Mg(DS)2micelles. These values correspond to field strengths of 4.1 X lo6and 5.0 X lo6 V/cm, respectively. Although these field strengths are not impossibly high, it is to be noted that a reduced AET value for SDS and Mg(DS)2micelles could also be due in part to interactions of the solubilized TEMPO with the high local concentrations of counterions expected for these charged micelles.z* If the local counterion concentration is assumed to be about 3 m for SDS, a value consistent with some considerations p r e ~ e n t e d , 2the ~ ~expected ~~ reduction in AET using the effect of NaCl on the ET values of TEMPO in water (Figure 2) is 0.36 kcal/mol. The remaining difference would then correspond to a field strength of 1.7 X lo6 V/cm. Similarly, in the case of Mg(DS)2, if the local counterion concentration at the surface is assumed to be one-half of 3 m, the calculated effect of the counterions is 0.46 kcal/mol (Figure 2). The remaining part of the charge effect would then correspond to a field of 1.9 X lo6 V/cm. These results indicate the importance of local field effects and suggest that such fields may be assessed by the approaches outlined. The changes in AET observed at different interfaces are of the order of thermal energy m d are likely to be significant for understanding specific effects of counterions and polar groups at interfaces of different charge types. The DeH value of ZWIG 12 is similar to that of CTAC. The zwitterionic head groups in this case are expected to produce a strong dipole field of the same direction as the electric field of CTAC. It seems that the dipole field at this interface is responsible for the lower Deffand higher A E T values of TEMPO. For the DPI probe also this effect has been found to be appre~iable.~'In the case of the phospholipid micelle P-LYSO, the Der and AETvalues are similar to those for OG. For this system the zwitterionic
TABLE 11: Comparison of Microenvironmental Polarities (D,ff) and AETof Several Nitroxides micellar system SDS Mg(DS), CTAC
5-
TEMPO
48 (2.03)'
OTEMPO
16-
NS NS
40 (2.47)b 47 46 (2.16)" 33 (3.00)Q 30 (3.14)b 33
45
TEMPA' 31 (3.18)' 31 (3.18)'
A & (kcal/mol) = ET(Water) - E ~ ( 0 ~ f refer f ) ; t o Table I. AET = ET(water) - ET(2Deff). ET water for OTEMPO is 69.13 kcal/mol. ET(Deff)calculated from the relation ET = 63.998 t 0.06650 established for hydroxylic solvents.z These data are judged to be somewhat AET(kcal/ less reliable than the data for TEMPO. mol) = ET(water) - ET(micel1ar system).
*
dipolar groups are separated from the hydrocarbon core by an intermediate layer of the glyceryl moiety. It seems that the proximity effect of the core is the primary microenvironmental feature in this case, the effect of the dipolar groups being small. This result is likely to be of general interest for many membrane lipids. Microenvironmental Effects and EffectivePolarities for Other Solubilized Nitroxides. The D,ff and A E T values for OTEMPO in SDS and CTAC have been estimated in a manner similar to that used for TEMPO. The results are given in Table 11. Because of lower values of K, for OTEMPO as compared to TEMPO and longer extrapolations involved in estimating the R, values, the D& values are less reliable than for TEMPO. The values are lower than those of TEMPO but the differences may not be very significant. The data in the accompanyingpaper indicate that the estimated fraction of solubilized OTEMPO at the interface is even higher than that of TEMPO.3 OTEMPO, however, has two polar moieties. The A E T and Deffdata suggest that the nitroxide group of OTEMPO is oriented toward the aqueous side of the micelle-water interface. Molecular models indicate that the OTEMPO can exist a t the interface in a sidewise orientation in which both polar groups are exposed to water to the same extent without a serious loss of hydrophobic interactions. The data are also consistent with a preferential orientation of the nitroxide group toward the aqueous side and are supported by some dodecane-water partition data presented in the accompanying papera3 When compared to model hydrocarbon compounds, the TEMPO data indicate that the free energy of transfer from dodecane to water becomes more negative by about 7.5 kcal/mol. The effect of the C = O group of OTEMPO, in comparison, decreases the free energy of transfer by about 2 kcal/mol. Thus, nitroxide groups are considerably more hydrophilic than C = O groups. For OTEMPO, if other factors are constant, a 100 to 1preferential orientation of the nitroxide group at the micellewater interface would involve a free energy difference of about 2.7 kcal/mol in the relative stabilization of the nitroxide group as opposed to the carbonyl group by hydrogen-bonding interactions. However, the possibility of sidewise orientation for some of the adsorbed OTEMPO molecules cannot be ruled out. This may explain the somewhat lower Deffvalues for OTEMPO as compared to TEMPO in view of the following arguments and observations for TEMPA+. In the case of TEMPA+, the Deffvalues were difficult to estimate for cationic or uncharged micellar systems because of very low micelle-water distribution coefficients. In SDS and Mg(D& micelles, however, because of very favorable electrostatic interactions, the cationic TEMPA+ is nearly completely solubilized. Thus, ,A, values characteristic of the solubilized state could be determined directly. The values were close to that of TEMPA+ in
Microenvironmental Effects on Nitroxkle Band Energies methanol. The Defland AET values of TEMPA’ in SDS and Mg(DS), (Table 11)were the same within experimental error. This similarity is consistent with the similarity of the Deffdata of TEMPO in these two systems. The Deff values of TEMPA+ are significantly lower than those of TEMPO and OTEMPO; Le., AET values are higher, even though, because of the ionic nature of TEMPA+, exclusive interfacial location of the nitroxide cation as a whole is expected, and the “dissolved state” should not be significant. Moreover, the charged group of TEMPA+ must reside in the aqueous part. The Deffvalue, however, still indicatea a relatively high polarity of the microenvironment of the nitroxide group a t the opposite end. Presumably TEMPA+ at the interface assumes an orientation in which the charged group, which must be located in the aqueous side of the interface, and the polar nitroxide group at the opposite end are both exposed to water so that the nitroxide dipole is parallel to the interface. This sidewise orientation is not likely to promote hydrogen-bonding interactions as much as the expected perpendicular orientation of TEMPO because of lower effective gN values. Thus, an increase in AET for TEMPA+ could involve this orientation effect as well as some contributions of orientations in which the nitroxide group is buried in the hydrocarbon core. It should be emphasized that differences in the net hydrophobic interactions for a typical group such as a CH2 group associated with complete transfer from water to a hydrocarbon, as in partition experiments, and adsorption to a hydrocarbon-water interface from water are of the order of 50 cal/moP so that the stronger hydrogen-bonding interactions are expected to play dominant roles in determining adsorption to the interface from the hydrocarbon core and relative orientations at interfaces. In the case of the long-chain systems 5NS and 16NS, low solubilities in water and dodecane precluded spectral measurements in these solvents. Because of their low solubility in water, however, these substances could be assumed to be completely solubilized in 0.3 M SDS and 0.2 M CTAC. The A- values in these systems were close to the ,A, values in methanol. When small corrections for small differences were made by assuming that the variation of A- with D is similar to that of either TEMPO or OTEMPO, the Deffvalues of 5NS and 16NS in SDS were 47 and 45, respectively (Table 11). The Deffvalue of 5NS in CTAC was 33. These values are very similar to those of TEMPO, indicating that the nitroxide groups of solubilized 5NS and 16NS are almost exclusively in the micelle-water interface region. Here again, it seems that the strong hydrogen-bonding interactions of the nitroxide groups confer upon them a pronounced tendency to seek the interface. This result is consistent with some reported results based on ESR data which indicate that the nitroxide portion of the methyl ester of stearic acid labeled in the 12 position experiences a highly polar microenvironment when solubilized in SDS micellesan The virtually identical A- values for 5NS and 16NS solubilized in SDS micelles are also in accord with the nearly identical values of the hyperfine splitting constants of these two labels in 0.35 M SDS reported by Yoshioka.4‘ These AN values also indicate a polar microenvironmentin a qualitative manner. Yoshioka interpreted his results differently, however. He assumed that the nitroxide group of 16NS in SDS micelles (46) Mukerjee, P. Adu. Colloid Interface Sci. 1967, 1, 241. (47) Yoshioka, H.J.Am. Chem. SOC.1979, 101, 28.
The Journal of Physical Chemistry, Vol. 86, No. 16, 1982 3205
is located in the hydrocarbon core and concluded that the polar microenvironment is produced by the presence of water molecules in the hydrocarbon core. This latter picture of the micellar interior has been shown to be inconsistent with a number of experimental observations and theoretical expectations based on the equilibrium solubility of water in hydrocarbon^.^^^^^^^^ I t appears to be unnecessary for explaining the observed facts. Indeed, this picture would require that in anionic micelles, the cationic species TEMPA+, expected to be completely at the interface for electrostatic reasons, has a microenvironment which is less wet than that of the nonionic TEMPO. It is interesting to note that monolayer studies at the airwater interface of nitroxide derivatives of stearic acid indicate strongly that the molecules are attached to water at the interface by both polar groups, namely, the acid end and the nitroxide moiety,50in agreement with the present interpretation. Implications for Spectroscopic Probe Studies of Lipid Assemblies. In conclusion some remarks regarding the use of nitroxides as spectroscopic probes of lipid assemblies are in order. These remarks are also generally applicable to numerous other spectroscopic probes currently being used to study lipid assemblies. It appears that in small or thin lipid assemblies such as micelles, and possibly membranes, where interfacial activity effects are magnified enormously by the very high surface-to-volumeratios,3J6J7 any assumption of a homogeneous distribution of even slightly polar molecules or molecules containing polar moieties in the hydrocarbon part is extremely hazardous. Any spectroscopic sensor of any microenvironmental property such as polarity or fluidity of lipid assemblies should be examined carefully with respect to favored locations, distributions, and orientations of the sensor molecules. The same remarks apply to sensor moieties. Thus, for example, attaching a probe in the middle of the chain of a long-chain amphipathic molecule does not assure that the probe will be buried in a lipid assembly. With respect to more detailed interpretation of polarity results, an estimated polarity intermediate between water and hydrocarbons does not necessarily indicate a mixture of water and hydrocarbons inside a lipid assembly. Indeed, such an intermediate polarity could arise from an exclusively interfacial location of the sensor moiety because of the inherent dielectric asymmetry of lipid-water interfaces. On the other hand, aside from their probe aspects, the phenomena investigated here are of independent interest and are of prime importance in understanding the interactions of polar molecules with lipid assemblies, and the nature of solubilization and distribution of solubilized species in lipid assemblies including physiological systems. The accompanying paper shows the relation of these microenvironmentalstudies on nitroxides with micelle-water and dodecane-water distribution equilibria and interfacial activities of n i t r ~ x i d e s . ~
Acknowledgment. This work was supported in part by the Public Health Service Research Grant GM-26078 and a fellowship to R.A.P. from the American Foundation of Pharmaceutical Education. We are grateful to a referee for many suggestions for a clearer presentation. (48) Mukerjee, P.; Mysels, K. J. ACS Symp. Ser. 1975, No. 9, 239. (49) Ulmus, J.; Lindman, B. J. Phys. Chen. 1981,85,4131. (50) Cadenhead, D. A.; Muller-Landau,F. Adu. Chem. Ser. 1975, No. 144, 294.
