formation of premicellar clusters of 2-p...

Langmuir 1992,8, 1271-1277 1271

Formation of Premicellar Clusters of 2-p-Toluidinonaphthalene-6-sulfonate with Cationic

Detergents Shufang Niu, Karical R. Gopidas, and Nicholas J. Turro*

Department of Chemistry, Columbia University, New York, New York 10027

Gavriella Gabor

Israel Institute for Biological Research, Ness-Ziona 74500, Israel

Received November 5, 1991. I n Final Form: February 10, 1992

2-p-Toluidinonaphthalene-6-sulfonate (TNS), a well-known fluorescent probe, forms premicellar aggregates with detergents such as cetyltrimethylammonium bromide (CTAB) at concentrations just below their critical micelle concentration. A narrow fluorescent band, very small Stokes shift, and very high quantum yield are the characteristics of the hydrophobic environment provided by the premicellar aggregates. Studies of the temperature dependence of fluorescence indicated that these species exist in solution as a separate microheterogeneous phase. These results are corroborated by results obtained from fluorescence polarization studies. The length of the alkyl chain is very critical in this case, and no such phenomenon is observed when the chain length is less than 14 carbon atoms. We propose that these species are formed by the enwrapment of TNS by the alkyl chains of CTAB molecules.

Introduction Simple surfactants such as sodium dodecyl sulfate (SDS)

and cetyltrimethylammonium bromide (CTAB), when dissolved in water, result in the formation of aggregates or micelles, if the concentration of the surfactants in solution is above the critical micelle concentration (cmc). For CTAB at room temperature in water containing no added electrolyte, the cmc is 9.2 X M and the aggregation number is ca. 100.' The structure of these micelles depends on such factors as the temperature, ionic strength of the solution, and the concentration of the surfactant. The understanding, however, is that, at concentrations above the cmc and below ca. 50 mM, the CTAB micelles are roughly spherical in shape. At concentrations below the cmc, the surfactant remains largely in the mon- omeric form. Ionic micelles have been used extensively as mediators for chemical reactions, and a large number of them have been probed by photophysical methods. However, studies of these surfactant solutions below their cmc are scarce. There are a few recent reports about the formation of premicellar aggregates and their ability to mediate reactions involving charged species.2 For example, Holzwarth et al. studied the rate of complex formation between metal ions such as Ni2+ and Mn2+ with a pyridine derivative in aqueous SDS solutions and found a considerable rate enhancement of complex formation below the cmc.2d Atherton et al. observed that divalent metal ions such as Zn2+, Ca2+, and Mg2+ forms clusters with submicellar SDS. Quenching of the singlet excited state of ethidium ions in these clusters by ground-state ethidium ions was found to be considerably higher in the premicellar aggregates.% Dye-induced aggregate formation in the premicellar region of SDS solution has been observed by Bax-

(1) Kalyanasundaram, K. Photochemistry in Microheterogeneous Systems; Academic Press: New York, 1987.

(2) (a) Baxendale, J. H.; Rodgers, M. A. J. Chem. Phys. Lett . 1980,72, 424. (b) Baxendale, J. H.; Rodgers, M. A. J. J. Phys. Chem. 1982,86, 4906. (c) Bruhn, H.; Holzwarth, J. Ber. Bunsen-Ges. Phys. Chem. 1978, 82,1011. (d) Holzwarth, J.; Knoche, W.; Robinson, B. H. Ber. Bunsen- Ges. Phys. Chem. 1978.82, 1001. (e) Sato, H.; Kawasaki, M.; Kasatani, K. J. Photochem. 1981,17, 243. (0 Atherton, S. J.; Dymond, C. M. G. J. Phys. Chem. 1989,93,6809. (g) Reitz, G. A.; Dressick, W. J.; Demas, J. N.; DeGraff, B. A.; Stephens, E. M. J. Am. Chem. SOC. 1988,110,5051.

0743-7463/9212408-1271$03.00/0

endale et a1.2a*b and also by Sato et aLZe In these studies it was observed that the rate of electron and energy-transfer reactions was considerably enhanced in the premicellar region. 2-p-Toluidinonaphthalene-6-sulfonate (TNS) is a well-

known fluorescent probe for study of the hydrophobic sites and conformational changes of protein^.^ The quantum yield of fluorescence of TNS and related compounds is very much higher in nonpolar than in polar solvents. Also the emission spectrum in a nonpolar solvent is blue shifted relative to the spectrum in polar ~olvents .~ The present report concerns the formation of aggregates between negatively charged TNS ions and positively charged CTAB molecules below the cmc as studied by the steady-state absorption and emission spectroscopies, steady-state fluorescence polarization measurements, and also fluorescence lifetime measurements. We present evidence that, in the premicellar region, CTAB molecules form aggregates which provide a very strong hydrophobic environment for TNS. This affects the absorption and emission properties of TNS considerably.

Experimental Section TNS potassium salt purchased from Aldrich was recrystal-

lized from methanol. CTAB, CTAC, and all other detergents were purchased from Eastman Kodak and were recrystallized from ethanol or ethanollether mixtures. Fresh solutions of TNS and CTAB were prepared for each experiment, and the measurements were performed approximately l h after the mixing of the reagents.

Absorption spectra were recorded on Cary 15 and Perkin- Elmer Model 559 UV/vis spectrophotometers. Fluorescence spectra were recorded on Hitachi Perkin-Elmer MPF 2A or LS-5 spectrofluorometers. Fluorescence polarization experiments were performed using a LS-50 spectrofluorometer with a computer- controlled polarization accessory. Relative quantum yields were measured using a solution of quinine sulfate in 0.1 N sulfuric

(3) Brand, L.; Gohlke, J. R. Annu. Reu. Biochem. 1972,41,843. (4) (a) McClure, W. C.; Edelman, G. M. Biochemistry 1966,5, 1908.

(b) Styrer, L. Science 1968,162,526. (c) Seliskar, C. J.; Brand, L. J. Am. Chem. SOC. 1971, 93, 5405. (d) Seliskar, C. J.; Brand, L. J. Am. Chem. SOC. 1971,93,5414. (e) Chiang, H.-C.; Lukton, A. J. Phys. Chem. 1975, 79, 1935. (0 Shinitzky, M. Zsr. J. Chem. 1974, 12, 879.

0 1992 American Chemical Society

1272 Langmuir, Vol. 8, No. 5, 1992

;..c , . . .

. .

Niu et al.

1 0.1s-

0)

C 0 e In $ 0.12-

0.06 -

0.0 , , I I 1

Wavelength, nm - 300 350 400 4 50

Figure 1. Absorption spectra of 1.25 X M TNS in the presence of various amounts of CTAB: (a) [CTAB] = 1.25 X 10-5 M (low CTAB); (b) [CTAB] = 1.25 X 10-3 M (micellar); (c) [CTAB] = 5 X M (premicellar).

acid (@ = 0.54).6 Lifetime measurements were made on a time- correlated single-photon counting system equipped with a Ed- inborough 199F nanosecond flash lamp, Ortec electronics, and a TN 1710 multichannel analyzer.

Results and Discussion Steady-State Experiments. Addition of CTAB to an

aqueous solution of TNS leads to significant changes in the absorption spectrum of TNS, the changes being dependent on the concentrations of CTAB. The absorption of 1.25 X 10-5 M TNS in the presence of various amounts of CTAB is shown in Figure 1. It is clear from Figure 1 that the shape and extinction coefficient of the lowest energy absorption band vary considerably with the amount of CTAB present in the system. For the purpose of discussion we classify the concentration of CTAB into three different ranges, lower range ([CTABI = <1 X M), premicellar range ([CTABI = 2 X l O - 4 t o 9 X M), and micellar range ([CTAB] = >1.1 X M). In the lower range of [CTAB], the lowest energy absorption shows a maximum a t 360 nm. In the premicellar range of CTAB, the extinction coefficient of this absorption increases and another equally intense band is observed at 370 nm. In the micellar range of CTAB concentration, the extinction of this band decreases again and the band maximum also blue shifts back to 356 nm. Even though the absorption maxima of TNS red shifts in the presence of premicellar CTAB, the tail of the lowest energy absorption (referred to as the "onset" of absorption) shows considerable blue shift (- 15 nm) compared to the lower and micellar regions of CTAB. This indicates that TNS is present in a relatively nonpolar environment when premicellar amounts of CTAB are present in solution. The onset of absorption and the extinction coefficients at different CTAB concentrations are given in Table I. These experiments have been

( 5 ) Parker, C. A.; Rees, W. T. Analyst 1962, 87, 83.

so3 Q DTCL3

4 0 0 440 480 5 20 560

Wavelength, nm-

Figure 2. Fluorescence spectra of TNS in various CTAB solutions: (a) premicellar (5 x 10-4 M); (b) low CTAB (1.25 X 10-5 M); (c) micellar CTAB (1.25 x M); (d) is the spectrum of TNS in ethanol.

Table I. Absorption Parameters of TNS in Aqueous CTAB Solutions

[CTAB] , M Xonsst, nm X,, nm e-, M-1 cm-' 0.0 400 356 5280 1.25 x 10-5 413 360 4680 5.00 x 10-4 398 370 9480

356 9360 1.25 x 10-3 412 356 4920

repeated with various TNS concentrations (2.2 X and 6.0 X

TNS is practically nonfluorescent in water, but it becomes fluorescent upon addition of small amounts of CTAB. The fluorescence spectra of TNS in the presence of various amounts of CTAB are shown in Figure 2. Since the absorption showed some difference due to the presence of CTAB, the samples were excited at 336 nm and the optical density of the solutions was matched a t this wavelength. It was observed that, in the lower range of CTAB, the fluorescence exhibits a maximum of 435 nm which is close to that reported for ita fluorescence in ethanol. When [CTAB] is in the premicellar range, the fluorescence shows a considerable intensity enhancement and a blue shift to 398 nm. The 398-nm band is much narrower than the 435-nm band. The Stokes shift in this case is much smaller than that observed in other solutions. When [CTABI is present in the micellar range, the fluorescence band again red shifts to 444 nm. This red shift is accompanied by a decrease in the intensity and broadening of the band. It is to be noted here that the band maximum shifts to 444 nm at a concentration of CTAB which is slightly higher than the cmc.

Since TNS is used in very low concentrations (- 10-5 M), any effect of TNS (such as ionic strength) on the cmc of CTAB is negligble. Plots of the variation in intensity, band width, and maximum against CTAB concentration are shown in Figure 3a-c. The various fluorescence parameters that are affected by the presence of CTAB are also summarized in Table 11. The luminescence intensities of these solutions varied slightly if they were kept for several hours. This effect was most prominent for solutions

M) with similar results.

Premicellar Clusters of TNS with Cationic Detergents

(3al 460

2 A

420-

-6 - 5 -9 -3 - 2 - 1 LOG (CTAB) +

Langmuir, Vol. 8, No. 5, 1992 1273

s X U I x

- 6 - 5 - 4 -3 -2 - 1 LOG (CTAB) -e

Figure 3. Various fluorescence parameters against CTAB concentration: (a) fluorescence maximum VB [CTAB]; (b) fluorescence band width vs [CTAB]; (c) maximum intensity vs [CTAB]; (d) fluorescence maximum vs [CTAB] in the presence of 0.1 M KCl.

Table 11. Fluorescence Parameters of TNS in Aqueous CTAB Solutions.

t CTABl , M A,, nm v , cm-1 W , cm-1 @ T , ns 0.0 (ice) 4556 ~ 0 . 5 ~ 0.0 (water) 0.0008c 1.25 x 10-5 435 5100 3000h 100 0.17 12.4 5.00X 10"' 404 2780 2600h 100 0.55 4.2 1.25 x 10-3 444 5260 3600* 100 0.22 5.2

from ref 4a. 4 v = Stokes shift,w = half-bandwidth. b Takenfromref4b. Taken

in the lower ranges of [CTAB] where the band maximum changes from 435 to 404 nm if the solution was kept in the dark for 2 days. The fluorescence excitation spectra in all these cases were very similar to the corresponding absorption spectra.

Fluorescence quantum yields of TNS solutions in the presence of various amounts of CTAB were also measured. These measurements were made relative to quinine sulfate in 0.1 N sulfuric acid. The quantum yield observed in premicellar CTAB was slightly higher than that observed in ethanol and considerably higher than that observed in the lower and micellar concentrations of CTAB. The quantum yield values are given in Table 11.

In an attempt to elucidate the reasons for the abnormal fluorescence behavior we have examined the effect of the addition of a salt (KC1) to solutions of TNS containing various amounts of CTAB. The result is shown in Figure 3d where the A, is plotted against log [CTAB]. It can be seen from Figure 3d that the shift in the fluorescence maximum occurs at a much lower concentration of CTAB. When KC1 is present at 0.1 M, the premicellar range extends from 8 X lo+ to 4 X 10-4 M of CTAB which is much broader than that observed in the absence of salt.

The behavior of TNS in the presence of several other detergents was also investigated. The results of these investigations are shown in Figure 4. It can be observed from Figure 4a,b that cetyltrimethylammonium chloride (CTAC, C-16 cationic detergent) and tetradecyltrimeth- ylammonium bromide (TDTAB, (2-14 cationic detergent) exhibit similar effects in aqueous solutions containing

TNS. However, no such effect was observed when TNS was present in aqueous solutions of dodecyltrimethylam- monium bromide (DDTAB, (2-12 cationic detergent) or decyltrimethylammonium bromide (DTAB, C-10 cationic detergent). Anionic micelles (SDS) and neutral micelles (TX-100) also did not produce any such effect. The results observed in these cases are shown in Figure 4c.

In an attempt to interpret the remarkable blue shift observed in premicellar solutions of CTAB, the effect of temperature on the fluorescence was studied in this region. In 1.25 X 10-6 M TNS solution containing 5 X lo-" M CTAB, the fluorescence intensity is reduced upon heating. Thereafter, the fluorescence spectrum showed drastic changes for every degree rise in temperature. The dependence of fluorescence intensity on temperature is shown in Figures 5 and 6. The variation of A, with temperature is shown in the inset of Figure 6. The fluorescence excitation spectra also showed considerable changes in this range of temperature. The intensity and maximum of these bands decreased and reached a stable state at 50 OC. These results suggest that the nature of the absorbing species undergoes a drastic phase change around 50 "C.

On the structure of the TNS-CTAB molecular aggregates, fluorescence polarization experiments were performed with TNS in the presence of various amounts of CTAB. The temperature dependence of polarization was also measured in the premicellar concentration of CTAB. The results of these studies are shown in Table 111. The dependence of polarization on temperature is shown in Figure 7. It can be noted from Table I11 that the polarization observed was much higher in the premicellar region, indicating that TNS is present in a more rigid environment in this case. The dependence of polarization on temperature (Figure 7) indicates that a phase transition is occurring in TNS solutions containing premicellar concentrations of CTAB around 45 "C.

Lifetime Measurements. The fluorescence lifetime of TNS in the presence of various amounts of CTAB is also given in Table 11. The fluorescence follows single exponential decay kinetics in all these cases. In ethanol

1274 Langmuir, Vol. 8, No. 5, 1992 Niu et al.

I X'

x A

-6 - 5 -4 -3 -2 -1 LOG (CTAC)-)

a x- 4 r

x 420 ::m 4 0 0 -6 -5 LOG(T0TAB)- -4 -3 -2 -1

VI z W t. z W

t

I

2 t J W a

4

3

2

1

0

-6 -5 - 4 -3 -2 -1 0

LOG (DETERGENT)

Figure 4. Fluorescence parameters in the presence of other detergents: (a) fluorescence maximum v8 [CTAC]; (b) fluorescence maximum vs [TDTAB]; (c) fluorescence intensity vs [detergent]: (H, TX-100; 0, DDTAB; 0, DTAB; 0, SDS).

- 5-

- l - 4-

J - %

In e .- ; 3- - C

u) .- .- E w 2- 0)

.- e - o 0) - a 1-

Wovelength,nm - Figure 5. Dependence of fluorescence on temperature of a solution of TNS (1.25 X M) and 5 X 10"' M CTAB: (a) at 30 OC; (b) at 40 OC ; (c) at 45 OC; (d) at 50 OC.

TNS has a lifetime of 12 ns which is close to the value calculated by Seliskar et al. (14 n ~ ) . ~ * In the presence of low [CTAB], TNS has a lifetime very similar to that in ethanol. However, in the premicellar and micellar ranges of [CTAB], the lifetime is reduced to 4 and 5 ns, respectively. Discussions. The absorption and emission charac-

teristics of TNS and related molecules have been studied in detail.4cvd It is well established that the fluorescence energies, bandwidths, and quantum yields of the (N-aryl- amino)naphthalenesulfonates show a marked dependence on the dielectric constant of the solvent medium a t 300 K and that these parameters correlate very well with the empirical solvent scale proposed by Koso~er .~a We shall consider the changes in absorption and emission spectra separately.

Absorption. The spectroscopic parameters of the two lowest energy singlet-singlet transitions in aminonaph- thalenesulfonate derivatives are similar to those observed for aniline and its derivatives. The low-energy electronic transitions in aniline have been interpreted in terms of a 'composite molecule" approach.6 In this theory, aromatic hydrocarbons having strongly conjugating groups are

(6) (a) Tasaki, 1.; Wantanabe, R.; Sandlin, R.; Carnay, L. Proc. Natl. Acad. Sci. U S A . 1969,62,612. (b) Murrel, J. N.Proc.Phys. Soc., London, Sect. A. 1955,68, 969. (c) Longuet-Higgins, H. C.; Murrel, J. N. Proc. Phys. SOC., London, Sect. A. 1955,68, 601.

: - T I 430 ::I, A,, , , , , I 400

20 x, 40 so so m 80 Temperalure t

Wovelength, nm - Figure 6. Dependence of fluorescence on temperature of a solution of TNS (1.25 X 10-5 M) and 5 X 10-4 M CTAB in the 40-60 OC range: (a) at 40 OC; (b) at 45 OC; (c) at 46 OC; (d) at 48 "C; (e) at 50 OC; (f) at 60 OC. The inset shows a plot of A, against temperature.

Table 111. Temperature Dependence of Fluorescence Polarization in Aaueous CTAB Solutions

[CTABI, M temp, O C polarizn [CTAB], M temp, O C polarizn 1.25 X 25 0.186 5.00 X lo-' 45 0.136 1.25 X 25 0.162 5.00 X lo-' 47 0.080 5.00 X 10"' 25 0.480 5.00X lo-' 50 0.075 5.00 X 35 0.437 5.00 X 10-4 60 0.068 5.00 x 10-4 40 0.395 treated as being made up of two component molecules. The electronic states of the whole molecule would then be some combination of the local states of both parts plus a certain percentage of electron-transfer states between the two components. Kasha et al.' suggested that the lowest energy transition in these molecules has intramolecular charge transfer character and referred to this as a* - 1 transition. "1" orbitals are lone pair orbitals which can bond relative to the A electron system of the molecule, and the extent of bonding depends on the angle of twist of the axis of the 1 orbital relative to that of the A orbital of the attached aromatic ring. Both n - A* and 1 - a, transitions have some common characteristics. In view of these earlier findings we try to explain the changes noted in the absorption spectra of TNS in the presence of various amounts of CTAB.

(7) Kaeha, M.; Rawls, H. R. Photochem. Photobiol. 1968, 7,561.

Remicellar Clusters of TNS with Cationic Detergents

O I \ ' . ' . ' . 1 Langmuir, Vol. 8, No. 5, 1992 1275

relaxation would, therefore, be expected to reduce the singlet-triplet split, facilitating intersystem crossing and thereby quenching fluorescence.lo

AlthoughTNS is nonfluorescent in water, upon addition of small amounts of CTAB, fluorescence is observed with Am, 435 nm. This maximum is slightly red shifted compared to that in ethanol. In a discussion of environ- mental effects on the fluorescence of TNS analogues Ainsworth et. distinguish between the terms "hydrophobic" and "nonpolar". They argue that the proximity of polar residues is a necessary but not sufficient condition to produce a red shift in the emission. For example, TNS is nonfluorescent in water but is fluorescent in ice.4b This means that in water the polar residues must also be able to relax around the TNS molecule in the few nanoseconds prior to emission. It is more appropriate under these conditions to suggest that the red shift is related to the presence of mobile dipoles. In aqueous solution TNS molecules are surrounded by polar water molecules. Upon addition of small amounts of CTAB (low concentration region) the TNS molecules are increasingly surrounded by nonpolar residues of CTAB which decreases the overall polarity and dielectric constant of the solution. The depletion of "mobile dipoles" (water) around TNS leads to the fluorescence observed in this case.

In the premicellar range of CTAB the emission maximum blue shifts to 404 nm. This can be explained by a hydrophobic protection of TNS molecules by CTAB. The relatively small Stokes shift indicates that no geometry change occurs in the excited state and also that solvent relaxation of the excited state is not important. The dependence of fluorescence excitation and emission intensity and shape on temperature is evidence enough for the hydrophobic association of TNS and CTAB in this range. An increase in temperature disrupts the hydrophobic cavity and releases the TNS into the water phase. The rupture of the surfactant cavity occurs on a very narrow range of temperature and resembles a phase transition as judged from the changes in the fluorescence spectrum. The dependence of Am, on temperature (Figure 6, inset) is clearly suggestive of a phase change in this region. This prompted us to believe that, in aqueous solutions of TNS containing premicellar amounts of CTAB, TNS molecules reside in a hydrophobic cavity formed by the CTAB molecules and that the changes noted in the absorption and emission spectra of TNS in this region are due to the hydrophobic environment experienced by the molecule.

It was observed that the premicellar association of detergents in the presence of TNS is measurable only when the chain length of the cationic detergents is 14 atoms or greater. This result shows clearly that the chain length is very critical in the formation of a hydrophobic cavity which we propose for the premicellar aggregates. The question arises as to whether interactions other than hydrophobic interactions are important in the formation of the aggregates, because both hydrophobic and hydro- philic effects are known to influence TNS fluorescence.12 Although we believe ion pairing (Chart Ia) is important for the effects a t low concentration, since >25 detergent molecules are required per TNS molecule to produce measurable effects, we do not believe such interactions are significant in the premicellar region (Chart lb).

In the micellar range of CTAB concentrations, the TNS fluorescence is observed a t 444 nm which is red shifted

(10) Jackson, G.; Porter, G. h o c . R. SOC. London, A 1961,260, 13. (11) Ainsworth, S.; Flanagan, M. T. Biochem. Biophya. Acta 1969,

(12) Beyer, C. F.; Craig, L. C.; Gibbons, W. A. Biochemistry 1972,11, 194, 213.

4920.

0.4 t e 0

0.0 1 ' I

20 30 4 0 5 0 6 0 7 0

Temperature * C Figure 7. Dependence of fluorescence polarization on temperature. [TNS] = 1.0 X lC5 M, and [CTAB] = 5.0 X 1o-L M.

TNS did not exhibit any new absorption in the presence of CTAB compared to that in water. This rules out the formation of any charge transfer complex formation between TNS an CTAB molecules in the concentration ranges studied. The absorption spectra of TNS in water, a t a low concentration of CTAB and in aqueous micellar CTAB solution, are somewhat similar. The absorption characteristics of TNS in premicellar concentrations of CTAB, however, were very peculiar. The blue shift in the tail of the lowest energy absorption may be due to a specific nonpolar environment experienced by TNS in premicellar solutions. The large increase in the extinction coefficient for the 1 - a* transition, however, cannot be explained by anonpolar environment alone. It seems more probable that the oscillator strength of the transition is enhanced by geometric changes occurring in the TNS molecule. We propose that in the premicellar range TNS molecules are embedded in a cavity formed by CTAB molecules which drives the geometric change in the structure of TNS. When TNS is present at 1 X M, the enhancement of absorption is noticed from 2.5 X to 1 X 10-3 M of CTAB. This means that for every TNS molecule there are 25-100 CTAB molecules present in solution. Since the phenyl and naphthyl moieties of TNS molecule are very hydrophobic, they may associate with the hydrophobic tails of CTAB molecules. In this particular arrangement that 1 orbital of the N atom in TNS may assume a favorable geometry for the enhancement of overlap with the u orbital of naphthalene, thereby increasing the oscillator strength and extinction coefficient of the 1 .-+a, transition. Further evidence for the formation of premicellar aggregates will be presented below in the discussion of the results of the fluorescence part.

Fluorescence. Fluorescence data yielded further evidence for the formation of premicellar aggregates. I t was proposed earlier by Forstera that fluorescence of anili- nonaphthalenesulfonates is observed only when the two ring systems are planar. I t was also suggested that intramolecular rotation of the phenyl and naphthyl rings causes an internal loss of energy, thereby reducing the yield of fluorescence.9 Another explanation given for the nonfluorescent nature of TNS in water is that the dipole moment of the first excited triplet state of this molecule is much less than that of the singlet excited state. Solvent

(8) F6,mter, T. Natururissenschaffen 1946.33, 220. (9) (a) Weber, G.; Laurence, D. J. R. Biochem. J. 1964,56, xxxi. (b)

Oeter, G.; Niehijima, Y. J. Am. Chem. SOC. 1966, 78, 1581.

1276 Langmuir, Vol. 8, No. 5,1992

Chart I

(a)

n

- - n U

000 0 0 (b)

\ t u

= water molecules

( C ) 0 = bromide i o n s

relative to its value in ethanol. The transformation from premicellar to micellar occurs a t concentrations slightly above the normal cmc (1.1 X instead of 9.2 X M). This indicates that the premicellar aggregates are not loosely bound species and that reorganization of these into normal micelles requires some driving force. Upon going from the premicellar to micellar phase the location of the TNS molecule changes from "inside the cavity" to "outside the micelle". Since the naphthalene chromophore of the TNS molecule carries a negatively charged sulfonate group, this part of the molecule is forced to reside on the outside of the positively charged CTAB micelles. In such a con- figuration the N-p-tolyl group of TNS is buried inside the micelle and the naphthalene chromophore is partly exposed to the water molecules and counterions present in the Stern layer of the micelle. This leads to a red shift in the fluorescence of TNS in the micellar region compared to the other two regions. The 435-nm peak in solutions of low CTAB concentrations is slightly blue shifted as compared to the 444-nm peak emitted by the micellized TNS molecule, indicating the less polar microenviron- ment of the former. We assume that it originates in a precursor of the premicellar aggregate. The slow formation of the 398-nm peak from the 435-nm peak at very low CTAB concentrations in the dark substantiates this interpretation.

The quantum yield of fluorescence and the fluorescence lifetime of TNS in CTAB solutions are also dependent on the concentration of CTAB (see Table 11). In a study of the dependence of the quantum yield of fluorescence of TNS in different solvents, McClure et aL4a observed that the quantum yield increased in the order methanol < ethanol < propanol. (Because of solubility problems, the quantum yield of TNS could not be determined in hydrocarbon solvents). Clearly the hydrophobicity of the alkyl chain plays a dominant role in the quantum yield of fluorescence. In the present case the highest quantum yield was observed for TNS in the premicellar region. This substantiates our conclusion that in this region TNS is present in hydrophobic aggregates.

Fluorescence polarization studies also clearly support the existence of different microheterogeneous phases.

Niu et al.

When CTAB is present in low concentrations, the observed polarization is 0.186. We were unable to compare this value to that in the absence of CTAB because TNS is nonfluorescent in water. This value, however, was higher than that obtained by Chiang et al.4e for TNS in SDS micelles near its cmc. When [CTAB] is above its cmc, the value of polarization decreased to 0.162 which is similar to that observed in SDS micelles.& These results indicate that even in very low concentrations, CTAB associates with TNS which reduces the molecular rotation of TNS. When CTAB is present in the premicellar range, the observed polarization is very high. Chiang et al.& observed earlier that the polarization of TNS in the presence of SDS increases considerably when salt is present in the system. This was attributed to the rigidity of the micelle due to the screening effect of the salt. In the present case the enhancement of polarization observed was much higher compared to that of the SDS-salt system. This corrob- orates our contention that the premicellar aggregates formed between TNS and CTAB are very rigid and hence restrict the motion of the TNS molecules. The fact that the polarization in this region showed a rapid decrease around 45 "C indicates the rupture of the premicellar cavity a t this temperature and release of the TNS molecules into the aqueous phase.

Lifetime Measurements. Luminescence lifetimes of probes such as tris(bipyridyl)ruthenium(II) dichloride (Ru- (bpy)32+) and ethidium bromide have been measured in the low, premicellar and micellar concentrations of SDS by earlier researchers.%bf For Ru(bpy)Q2+ in aqueous SDS, Baxendale et al.2a observed that in the low concentration region of SDS the probe decay was characterized by a fast component and a slow component, the latter being equal to the normal decay of the probe. As the concentration of SDS increases to the premicellar range, the fast component becomes faster and then disappears at concentrations above cmc. The fast component in this case was assigned to be due to triplet-triplet annihilation reactions occurring due to the multiple occupancy of Ru- ( b ~ y ) 3 ~ + in the premicellar clusters. These researchers proposed that the clusters contain 3-8 probe molecules. In another study, Atherton et al.2f showed that ethidium ions form clusters with submicellar SDS. Multiple occupancy of these clusters by ethidium ions leads to double exponential decays arising from the quenching of the excited singlet state by ground-state molecules.

The case we present here differs from these earlier reports in that the fluorescence lifetime of TNS was single exponential in the low, premicellar and micellar ranges of CTAB concentrations. Typically when a fluorescing molecule is micellized, the fluorescence lifetime increases. However, when TNS is micellized in CTAB, we see a lifetime decrease. We proposed earlier that, in micellar solutions of CTAB, TNS is positioned in the water-micelle interface, thereby partly exposing the naphthyl chromophore to water molecules and counterions. The water molecules can quench the fluorescence by increasing the intersystem crossing rate. The same effect can be achieved by bromide ions through a heavy atom effect. However, in the present case the contribution from the heavy atom effect is found to be negligible because the lifetimes were similar in CTAB and CTAC micellar solutions.

TNS in premicellar solutions of CTAB shows a lifetime which is much smaller than that in ethanol. We do not believe that this can be attributed to any aqueous quenching mechanisms because in this case the TNS molecules are encapsulated by the hydrophobic tails of CTAB protected from water molecules. We seek an explanation

Remicellar Clusters of TNS with Cationic Detergents

of this result that also explains the increase in the extinction coefficient of absorption in premicellar solutions. An estimate of the radiative lifetime of an emitting molecule may be obtained from the approximate rela- tionship 70 = 104/emm. When TNS is dissolved in premicellar solutions of CTAB, e- changes from -5000 to -9500 M-1 cm-1. Since emax and 70 are inversely related, we would expect a decrease in the fluorescence lifetime of the molecule. The contention here is that the TNS molecules we are dealing with in the low, premicellar and micellar concentrations of CTAB are not exactly the “same species”. TNS in the premicellar range has different electronic energy levels compared to the other cases, and they can be considered as geometrical isomers. Since these are different molecular entities, a comparison of the lifetimes is not warranted.

In light of the discussion above, we draw the following picture for the solubilization of TNS in aqueous solutions containing various amounts of CTAB. In the lower range of concentrations, both TNS and CTAB move around randomly in solution, but at any given time TNS molecules are exposed to some hydrophobic interactions provided by the alkyl chains of CTAB. The formation of ion pairs between TNS and CTAB is a real possibility under these conditions. In this arrangement the negatively charged sulfonate group of TNS remains close to the positively charged trimethylammonium head group of CTAB and the alkyl chain of CTAB folds back to provide a hydrophobic environment for the TNS molecule (Chart Ia). This reduces the effective polarity of the environment experienced by TNS and hence increases the quantum yield of luminescence compared to that in water. In the premicellar range of CTAB concentrations, clusters of CTAB lie around a given TNS molecule (TNS may be acting as a seed for cluster formation). The cavity thus provided by CTAB is very hydrophobic and very rigid (Chart Ib) so that it affects the geometry of the TNS molecule and hence ita absorption and emission characteristics. The lowest energy transition becomes more allowed, the fluorescence lifetime decreases, and the fluorescence quantum yield increases. This is analogous to the ob- servation of Reitz et al.2h that certain rhenium complexes exhibit a unique intramolecular perturbation of their excited-state manifold by alkyl chains connected to the complex. The degree of intramolecular fold back is a strong function of the chain length and alters the solvent environment around the excited portion of the molecule with a concomitant change in the state energies and decay paths. In the present case, the alkyl chains are provided by CTAB molecules, and this particular arrangement can be considered as a first or induced micellar phase for the system. The fact that this first micellar phase continues to exist even at concentrations above the normal cmc indicates that this phase is somewhat stable, and formation

Langmuir, Vol. 8, No. 5, 1992 1277

of the second or native micellar phase from this requires additional driving force. I t is to be mentioned here that the length of the alkyl chain is very important in this case also. Alkyl chains of less that 14 carbon atoms did not produce any premicellar effect. This prompted us to believe that the first micellarphase is rodlike or cylindrical in shape. In the fully stretched form the length of the TNS molecule will be about 14 A. This is about the length of a fully extended C-12 alkyl chain. The fact that premicellar aggregation is not observed in C-12 detergents indicates that the chain length must be larger than the length of a TNS molecule to give effective protection from polar residues. When [CTAB] reaches the second micellar range, rearrangement of the molecules occurs. Since the attraction between the positively charged micelles and negatively charged TNS is electrostatic in nature, TNS will be solubilized on the surface of the micelle with the sulfonate group projecting into the aqueous phase (Chart IC). This exposes the active chromophore (naphthyl) to water, resulting in the quenching of fluorescence by water (mobile dipole). This would result in a red shift and reduced quantum yield and lifetime for the fluorescence of TNS in micellar CTAB.

Conclusions Studies of the absorption and emission properties of

TNS in aqueous solutions containing different amounts of CTAB reveal the existence of different microheterogeneous phases in the solution. When CTAB is present in the lower concentration range, the TNS.molecules are most probably present as ion pairs in an environment which is less polar than water. If this sample is allowed to stand for sufficient time, the detergent molecules rearrange to form a cavity around TNS as is evident from ita emission spectrum. When CTAB molecules are present in premicellar concentrations, they wrap around TNS molecules, thereby providing a very hydrophobic cavity. TNS molecules may be acting as a seed for the formation of the cavity, and the cluster formed by TNS and CTAB can be considered as an induced micellar phase. The exact number of CTAB molecules that is needed for the formation of this phase and their precise arrangement are uncertain at this time. Finally, when the micelles are formed, the TNS molecules flip over from the inside of the premicellar cavity to the Stern layer and become partially exposed to the aqueous phase.

Acknowledgment. We thank the National Science Foundation, the Department of Energy, the Air Force Office of Scientific Research, and the Office of Naval Research for their generous support of this research.

Registry No. CTAB, 57-09-0; TNS potassium salt, 32752- 10-6.

formation of premicellar clusters of 2-p...

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