Photophysical investigation of rose bengal in aqueous sodium taurocholate solutions

J. Photochem. Photobiol. B: Biol., 17 (1993) 41-56

Photophysical investigation of rose bengal in aqueous sodium taurocholate solutions Alain

Seret+ and Albert

E+erimental (Received

Van de Vorst

Physics, University of Ltige, Institute of Physics, B5, Sart Tilman, B-4000 LiPge (Belgium) May 22, 1992; accepted

July 17, 1992)

Abstract The photophysics of rose bengal has been studied in aqueous sodium taurocholate solutions with or without 0.6 mol NaCl dmV3. The ground and first excited singlet states solubilize only in the aggregates formed by sodium taurocholate, whereas the first triplet state solubilizes both in the bulk phase and in the aggregates. A lower limit of the corresponding entry and exit rate constants k, and k, respectively has been estimated: k, > 5 x lo6 dm’ mol-’ s-’ (2x10’ dm3 mol-’ s-t with 0.6 mol NaCl dmm3) and k,>lO’ s-t. The triplet state as well as the semioxidized radical react with the taurocholate anion. The former reaction leads to the formation of semireduced dye. The latter is responsible for the detection of only semioxidized radicals which move freely in the aqueous phase. The results are compared with those obtained previously in aqueous sodium dodecyl sulphate solutions and the possible biological implication is discussed.

Keyrvords: Absorption, state.

bile salt, flash photolysis,

fluorescence,

1. Introduction Rose bengal (RB) is probably one of the most used synthetic dyes by photobiologists and photochemists. Large absorption in the visible, high triplet and singlet molecular oxygen quantum yields, and solubility in both water and moderately polar media are certainly among the reasons which explain this popularity [l]. The photophysics and the photochemistry of RB in homogeneous media have been largely investigated (for reviews see for example refs. 1 and 2). However, living matter is essentially a heterogeneous medium. The compartmentalization of the molecules which results from this heterogeneity has important implications on chemical reactions because, while some reactants are held apart, others are brought into close contact [3, 41. The existence of various microenvironments with different physicochemical characteristics can greatly influence dye photophysics which is generally environment dependent [5]. In order to elucidate the influence of the medium heterogeneity on the photochemistry and photophysics of dyes, simple models such as micelles, ‘Author

to whom correspondence

101 l-1344/93/$6.00

should be addressed.

radical,

rose bengal,

sodium

taurocholate,

triplet

reverse micelles or vesicles have been widely used [3]. Although they can be viewed as primitive, these models comprise the principal features of cellular membranes: two regions with distinct physicochemical properties that are separated by an interface whose electrical charge and length can be chosen by an appropriate selection of the amphiphile molecule [3, 41. In recent years, a lot of photophysical studies of RB and other halogenated fluorescein derivatives in micellar media have appeared [6-181. Most of them have been devoted to aqueous solutions of cationic surfactant where the attractive forces between the surfactant head group and the dianionic dye lead to the formation of premicelles, dye-rich micelles or singly occupied micelles depending on the detergent concentration 17, 8, 10, 13, 14, 171. In the singly occupied micelles, RB, whatever its electronic state, is bound strongly to the micellar surface and does not exit from the micelles to the bulk phase [7, 8, 171. Several studies have also investigated aqueous solutions of RB and an anionic surfactant, generally sodium dodecyl sulphate (NaDS) [S, 9, 15, 16, 181. In principle, the electrostatic repulsion between the anionic head group and the dianionic dyes should preclude any binding of the dye to

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48

A. Seret, A. Van de Vorst I Rose bengal in aqueous Na taurocholate

the micelles. However, the hydrophobic interaction is so large that the binding occurs [8, 9, 15, 161. Hence, ground state [8, 91 and semioxidized [15] RI3 were found to be exclusively solubilized in the NaDS micelles. In contrast, the RB triplet state exhibits a partitioning between the bulk phase and the micelles, reflecting a less hydrophobic character when the dye is excited in its triplet state [16]. The photosensitizing action of RB proceeds generally via a type II mechanism, i.e. energy transfer from dye triplet state to ground state molecular oxygen followed by an oxidative reaction between excited oxygen and a substrate [2]. Nevertheless, some recent reports give insight for an implication of radical mechanisms under special circumstances [19, 201. As the diffusion of excited species could be very limited in biological media because of the presence of numerous targets, knowledge of the solubilization site of the triplet state from which singlet oxygen is generated and of the radicals of RB is of prime interest. This point has been clearly highlighted in a recent work where the association of RB with the membrane of Gram-negative bacteria has been found to be a critical factor determining photosensitization efficacy [21]. The surfactants used in all the studies cited above are synthetic products. The bile salts, which are generated in the liver of mammals, can be considered as natural detergents [22, 231. Photophysical investigations in aqueous solutions of bile salts could be considered as a further step in order to bring the studies in simple heterogeneous systems closer to the biological reality. Surprisingly, studies of this kind are very scarce and, to our knowledge, have always been limited to the ground and first excited singlet states of hydrophobic chromophores [24-291. The present work was undertaken with the aim of investigating the photophysical properties of RB in sodium taurocholate (NaTC) aqueous solutions. Particular emphasis was placed on dye triplet state and semioxidized radical. Among the numerous existing bile salts, NaTC was chosen because its aggregation process is known to be not so sharp as those of synthetic detergents [25, 301. Hence, in aqueous solutions of NaTC, the transition region between pure monomeric solution and micellar solution is so large that it is very difficult to define a critical micelle concentration (CMC) [23, 25, 30, 311. Moreover, the aggregates are continuously changing with the NaTC concentration [25, 28, 291. This last point is of great importance in the case of RB because it was observed that small changes in NaDS micelles are

able to influence the partitioning of its triplet state between the micelles and the bulk phase [16]. The studies were generally performed in lo-2 mol NaOH dmp3 aqueous solutions in order to avoid dye protonation. Sometimes, 0.6 mol NaCl dme3 was added to the solutions because, at this NaCl concentration, the NaTC-H,O system is thought to be better defined, i.e. the polydispersity is lower and more reliable values of the mean aggregation number have been determined [25].

2. Experimental

details

NaTC was purchased from Calbiochem (La Jolla, CA; lot 910330). RB was supplied by Eastman Kodak (Rochester, NY). Careful purification of this dye was achieved by means of gel chromatography [32] on Sephadex G-25 (Pharmacia, Uppsala, Sweden). The ground state molar absorption coefficient of purified RB is lo5 dm3 mol - ’ cm- ’ at 548.5 nm in 10F2 mol NaOH dmp3. NaCl, NaOH and LFe(CN)6 were analytical grade products from Merck (Darmstadt, Germany). K,Fe(CN), was obtained from J. T. Baker (Deventer, The Netherlands). CuCl, and CaCl, were pro-analysis salts from Union Chimique Belge (Bruxelles, Belgium). Laboratory deionized and distilled water was used for all solution preparations. Stock solutions of NaTC were prepared fresh daily by dissolution of the solid in this water. Absorption spectra were recorded with a Perkin-Elmer 559 UV-visible spectrophotometer (Perkin-Elmer, Oak Brook, IL). Fluorescence measurements were made with an SLM-Aminco SPF500C spectrofluorimeter (SLM Instruments Inc., Urbana, IL). Fluorescence emission spectra were collected at an excitation wavelength of 500 nm and a 1 nm scanning interval with a 1 nm emission slit. These spectra were corrected for instrument response with use of the built-in correction factors. For emission intensity determinations, the absorbance of all the samples was 0.030 at 500 nm. The conventional flash photolysis apparatus has been described elsewhere [14, 151. Since these descriptions were published, the monitoring light source has been replaced by a 100 W tungsten-halogen lamp (Osram Xenophot 12 V, Berlin, Germany) powered by an Alpha Elettronica AL 626-S stabilized d.c. power supply (Alpha Elettronica, Parma, Italy). The outer jacket of the photolysis cell was filled with a liquid filter mixture of 0.21 mol CuCl, dmm3 and 1.06 mol CaCl, dmv3 that cuts off wavelengths below 400 nm and above 700 nm. When necessary, the samples were

A. Seret, A. Van de Vorst / Rose bengal in aqueous Na taurocholate

deoxygenated by saturating with nitrogen [16]. In these N,-saturated solutions, the flash energy was chosen to ensure total conversion of the dye to its triplet state [16]. Experimental uncertainty is 5% on the triplet half-life and 10% on the halflife of the radicals and all the maximum transient absorbances [15, 161.

3. Results

and discussion

3.1. Singlet states The absorption maximum, the fluorescence emission maximum, and the relative fluorescence intensity of RB are reported in Table 1 for aqueous solutions containing lo-* mol NaOH dmw3, 0 or 0.6 mol NaCl dmP3, and several NaTC concentrations. Without NaTC, the presence of 0.6 mol NaCl dme3 does not alter the fluorescence intensity of RB. The maximum absorbance of samples containing identical RB concentrations and, therefore, RB ground state absorption coefficients does not depend on NaTC concentration. No isosbestic point can be found between the absorption spectra recorded at different NaTC concentrations. Clearly, at NaTC concentrations higher than or equal to 1 mmol dme3 the absorption and emission maxima are shifted to the red and the fluorescence emission intensity is increased (Table 1). In the case of halofluoresceins, such modifications indicate a lowering of polarity and/or hydrogenbonding capabilities of their direct environment [l, 21. In aqueous surfactant solutions, it is due to the formation of micellar aggregates in which TABLE 1. Maximum absorption and fluorescence emission wavelengths hAb and h, respectively, and relative fluorescence intensity Is of rose bengal in aqueous solutions containing 10-a mol NaOH dme3 and various concentrations in NaTC and NaCl

[NaTC] (mmol dme3)

0 1 2 3 4 5 6 7.5 10 15 20 30

[NaCI] = 0 ‘+Ab

AR

@ml

(nm)

548 548.5 549 549.5 550 551.5 n.d. 554 555.5 558.5 559 560

567 569 569.5 570.5 570.5 571.5 n.d. 576 577 578 578.5 580

n.d., not determined.

[NaCI] = 0.6 mot dm-3 Ifl

1 1.07 1.14 1.21 1.23 1.33 n.d. 1.48 1.63 1.90 2.15 2.32

IfI $n)

$m)

548 550 551.5 554.5 557 559 560 560.5 561 n.d. 562 562.5

567 569.5 573 575 576 577 579 580 581 n.d. 581.5 582

1 1.16 1.29 1.52 1.91 2.07 2.47 2.62 3.01 n.d. 3.85 4.48

49

the dye can be solubilized [8, 181. Consequently, the results presented in Table 1 show that, above 1 mm01 dmw3, NaTC builds aggregates in which ground and first excited singlet states of RB can be solubilized. Counterion binding to NaTC micelles is nearly zero [22]. Therefore, the binding of dianionic RB to NaTC micelles results from a predominance of the hydrophobic interaction over the electrostatic repulsion. The same interpretation has been proposed to explain the solubilization of RB ground and first excited singlet states [8, 91 and RB semioxidized radical [15] in anionic micelles of NaDS. It is necessary to explain the progressive shift of maxima and the gradual increase in fluorescence intensity observed when increasing bile salt concentration. In NaDS micellar solutions, the increase in surfactant concentration leads to a progressive shift of the maxima and a gradual increase in fluorescence intensity [8, 331. Time-resolved fluorescence experiments have clearly shown that, above the CMC, ground and first excited singlet states of RB are exclusively solubilized in the NaDS micelles [9]. The gradual shift of maxima and the progressive increase in fluorescence intensity are thought to result from the decrease in hydrogenbonding capabilities of the micellar interface that follows the increase in NaDS concentration. Kamat and Fox [34] have claimed that the partition of RB between the aggregates and the bulk phase of a polymer-alcohol mixture results in a sharp shift of the absorption maximum and the presence of an isosbestic point between the spectra recorded at different polymer concentrations. The situation in aqueous NaTC solutions appears to be comparable with that encountered in aqueous NaDS solutions. Consequently, it can be inferred that RB ground and first excited singlet states are exclusively solubilized in NaTC aggregates and that the micellar environment of RB is becoming progressively less “aqueous like” between 1 and 30 mmol NaTC dmw3. Seemingly, the addition of 0.6 mol NaCl dme3 enhances the effect. This would explain why the red shifts and the intensity increase are larger in the presence of NaCl (Table 1). NaTC micelle growth has been observed on increasing the bile salt concentration or adding NaCl [23, 35, 361. Maybe, bigger NaTC micelles can offer a higher protection to RB towards water molecules by better surroundings of the dye with micellar matter. Another explanation could be that the micelle growth is followed by a partial dehydration as has been suggested for NaDS [15, 15, 37, 381. Whatever the correct interpretation,

A. Seret, A. Van de Vent I Rose bengal in aqueous Na taurocholate

50

the data presented here show that the solubilization site of RB singlet states in NaTC micellar aggregates is less hydrophilic in character at higher bile salt concentration or in the presence of 0.6 mol NaCl dme3. 3.2. Ttiplet state 3.2.1. Transient spectra

Flash photolysis of RB in N,-saturated aqueous solution containing lo-’ mol NaOH dm-3, 0.6 mol NaCl dme3 and 20 mm01 NaTC dme3 generated the transient-minus-singlet difference spectra presented in Fig. 1. Similar difference spectra have been recorded at different NaTC concentrations between 1 and 30 mm01 dmm3 and without NaCl. All the spectra exhibit a positive absorption from 400 to around 500 nm with two maxima at 425430 nm and 480 nm, followed by a depletion region, which corresponds to the lowest energy absorption band of RB ground state, and a positive absorption again from 595 to 680 nm. Previous flash photolysis and pulse radiolysis studies of RB in various media [16, 39-411 have assigned the

red band to the triplet state and the band with a maximum in the 420-430 nm region to the semireduced radical. Semioxidized RB is believed to exhibit an absorption maximum at 465470 nm [16, 19, 401. However, the decay kinetics of the absorption band with a maximum at 480 nm is very similar to that of the triplet state and great care must be taken in assigning this absorption band. In Section 3.3 it is shown that semioxidized RB absorbs effectively at these wavelengths in NaTC solutions. It is also explained why the absorbance of this radical cannot be detected in N2saturated solutions. The decays of the triplet state and the semireduced radical have been monitored at 600 nm and 425 nm respectively. In N,-saturated solutions, the initial height of the triplet signal does not depend on NaTC concentration. Since the flash energy is chosen to ensure total conversion of the dye to its triplet state (see Section 2) this observation means that the triplet-minus-ground state absorption coefficient is independent of NaTC concentration. 3.2.2. Solubilization site In aqueous solutions irradiated by visible light, production and deactivation of the RB triplet state proceed by the following pathways [16, 17, 411: ‘RB,, -%

10

3RBk7-

7.5

2 "0 5 30 us

3OPS lms 3OOGS

2.5 i

i\-3oous

Lm

C

Ems bh\ LOO

,!

450

LJ)O: ;

JlI-y-

600

650

X1nf-n) Fig. 1. Difference spectra of a N,-saturated aqueous solution containing 2.4 pmol RB dmm3, 20 mmol NaTC dm-‘, low2 mol NaOH drnm3 and 0.6 mol NaCl dmm3 recorded at different times after the flash excitation.

‘RB, 4

3RB

‘RB,

(1) (2)

3RB + 3RB -

3RB + ‘RBO

(3a)

3RB + 3RB -

2rRB0

(3b)

3RB+3RB-

RB’-+RB-+

3RB + ‘RB, -

2’RB,,

3RB+‘RB,-

RB-+RB’+

(4) (5)

(6) where ‘RBo, ‘RB, and 3RB denote ground, first excited singlet and first triplet states respectively of RB. The triplet-triplet annihilation reaction is written in the literature as either reaction (3a) (see for example ref. 16 and references cited therein) or reaction (3b) [17, 411. The formation of semireduced (RB-) and semioxidized (RB’+) radicals results only from the bimolecular quenching reactions (eqns. (4) and (6)) of the triplet state. Electron transfer reaction implicating the RB first excited singlet state does not occur, except in the presence of high concentrations (at least mmol dme3) of specific quenchers. For RB in basic aqueous NaTC solutions, the triplet half-life and the semireduced radical production yield, which is proportional to the maximum transient absorbance at the radical absorp-

A. Seret, A. Van de Vorst / Rose bengal in aqueous Na taurocholate

tion maximum [16], have been function of bile salt concentration Hexacyanoferrate(II1) is able triplet state both physically and

measured as a (Figs. 2 and 3). to quench RB chemically [16].

_.I_ _. -.

_.J_..

Oj

1

{aTC]( ds, mmol

3o

Fig. 2. Dependence of RB triplet half-life on NaTC concentration in N,-saturated aqueous solutions containing 10-r mol NaOH dm-’ and 2.4 (0, l), 4.8 (0, n) pmol RB dm-‘, with (0, I) or without (0, 0) 0.6 mol NaCl dm-‘.

51

The chemical reaction leads to the formation of semioxidized dye. Because of its triple negative charge and its hydrophilic character, Fe(CN),3is expected to be exclusively solubilized in the aqueous phase of anionic micelle solutions. The dependence of RB triplet half-life and semioxidized radical production yield on the hexacyanoferrate(II1) and NaTC concentration is illustrated in Figs. 4 and 5 respectively. These data show that, at concentrations higher than or equal to 1 mm01 dme3, NaTC hinders the reaction between RB triplet state and Fe(CN),3-. This observation indicates clearly that a partial solubilization of dye triplet state in micelles occurs above 1 mm01 NaTC dmm3. Now, it will be shown that the peculiar shape of the curves in Fig. 2 could be rationalized in terms of a competition between triplet-triplet annihilation and triplet reaction with taurocholate anion. We first focus on the following NaTC concentration range (called range 2 from here on): 3-15 mm01 dmP3 or 3-10 mm01 dmm3 in the presence of 0.6 mol NaCl dmm3. In this range, any increase in NaTC concentration is followed

30_ z. -.. _

a ,

0

-. _..

1

(a)

_.L.

5_

01 0

1 10 [NaTC](mmol

20 drr?)

Fig. 3. Variation in the yield of semireduced RB with NaTC concentration in NTsaturated aqueous solutions containing 10-r mot NaOH dm-’ and 2.4 (0, l), 4.8 (Cl, I) pmol RB dme3, with (a, n) or without (0, 0) 0.6 mol NaCl dm-‘.

(b)

b I 2

I L

[Fe(CN),3-](lf5mol

_

1. r-.

6

8

dmm3)

Fig. 4. Dependence of RB triplet half-life on Fe(CN),‘and NaTC concentrations in N,-saturated aqueous solutions containing 10-r mol NaOH dm- 3, 2.4 pmol RB dm-x and (a) 0 and (b) 0.6 mol NaCl dme3. NaTC concentrations are indicated on the plots and expressed in millimoles per cubic decimetre.

52

A. Seret, A. Van de Vorst I Rose bengal in aqueous Na taurocholate I

I

0

a A

(b) Fig. 5. Dependence of the yield of semioxidized RB on Fe(CN),3and NaTC concentrations in air-saturated aqueous solutions containing lo-’ mol NaOH dm-3, 2.4 pmol RB dmm3 and (a) 0 and (b) 0.6 mol NaCl dmm3. NaTC concentrations are indicated on the plots and expressed in millimoles per cubic decimetre.

by an increase in triplet half-life and a decrease in the difference between the triplet half-lives recorded for two different dye concentrations (Fig. 2). These observations are in line with the partial solubilization of RB triplet state in NaTC aggregates. Indeed, the incorporation of triplets in micelles restricts the dye-dye quenching (reactions (3)-(6)) w h’ic h is an important source of RB triplet state decay in flash photolysis experiments [16, 39, 411. We now turn our attention to the O-3 mm01 NaTC dme3 concentration range (range 1). Above 1 mm01 dmw3, the partial solubilization of RB triplet state in bile salt aggregates implies that triplet-triplet collisions are restricted. This restriction, if alone in determining triplet half-life

and radical production yield, would lead to an increase in triplet half-life, a reduction in the difference between the triplet half-lives recorded for two different dye concentrations and a decrease in the radical yield. The data presented in Figs. 2 and 3 do not corroborate this expectation. Particularly interesting are NaTC concentrations where a slight increase in semireduced radical production yield occurs (Fig. 3). They are also characterized by a decrease in triplet half-life (Fig. 2). Quenching of RB triplet by taurocholate ion that leads to the formation of semireduced RB could account for this observation. The last NaTC concentration range (range 3) to be examined extends from 15 to 30 mmol dme3 or from 10 to 30 mmol dmV3 in the presence of 0.6 mol NaCl dm-‘. In this range, the RB triplet half-life depends less and less on the concentration in dye (Fig. 2) or Fe(CN),3(Fig. 4). This higher protection of RB triplet state against the quenching mechanisms by the dye itself or Fe(CN),3indicates that more and more RB triplet states are incorporated in NaTC micelles. In the micelles, the triplet state remains probably able to react with a taurocholate anion. More RB triplet states solubilized in the micelles implies more reaction events with taurocholate ions. This could explain the trend of the triplet half-life to decrease slightly (Fig. 2) and the trend of the semireduced radical production yield to increase slightly (Fig. 3) in range 3. The most striking outcome of the above discussion is that, at concentrations higher than or equal to 1 mm01 dme3, there exist NaTC aggregates where the RB triplet state finds a solubilization site. The global structure of these aggregates is sensitive to NaTC concentration and the presence of NaCl[23,27,36,42]. In particular, the aggregate size seems to increase with NaTC and NaCl concentrations. However, the results presented here indicate that the NaTC micelles formed at low or high bile salt concentration and with or without NaCl are able to incorporate RB triplet state. Consequently, if 3RBM represents the RB triplet state solubilized in a micelle, the following equation can be written, regardless of the structure of the NaTC micelle (M). kS 3RBM = 3RB+M (7) k. 3.2.3. Entry and exit rate constants Because of the exclusive solubilization of the RB first excited singlet state in the micelles (uide supra), the intersystem crossing to the triplet state

A. Seret, A. Vun de Vorst / Rose bengal in aqueous Na taurocholate

occurs in the micelles, i.e. [3RB] =0 at zero time. Moreover, the reactions (5) and (6) should not be taken into account. Because the micelle mean occupancy number is far lower than unity (low dye-to-micelle concentration ratio), the dye-dye reactions in micelles can be neglected. Therefore, the triplet decay is governed by reactions (2)-(4) and (7), the unimolecular decay of micelle bound triplet (rate constant k,,) and, eventually, the triplet quenching by Fe(CN)63- (reaction rate constant k&. The corresponding kinetic equations are -

d[3;M1 = (k,+k,)[3RBM] -k,[M][3RB]

- F

(8)

= (k,[M] +kT + k,,[Fe(CN),3-])[3RB] + ,&J3RB]’ - kJ3RBM]

(9)

where k.,-r is the overall rate constant of reactions (3) and (4). The results presented in Fig. 5 were obtained during aerobic experiments. As calculated in ref. 16, the RB triplet lifetime is reduced to a few microseconds in air-saturated solutions. The increase in the yield of semioxidized radical with Fe(CN),3 - concentration shown in Fig. 5 indicates that, under these conditions, the RB triplet state is still quenched by Fe(CN),3-. As this quenching occurs only in the aqueous phase, and taking into account the initial condition ([3RB] =0), it can be concluded that the inverse of the exit rate constant k, is at least of the same order of magnitude as the aerobic lifetime of RB triplet state, i.e. k, > 10s S-l.

As shown in the preceding paragraph, the formation of 3RB is fast and occurs within about 10 ps. For other surfactants, k,M is close to k, [l7]. Therefore, on the assumption that this property subsists in NaTC, kTM = kT = 6700 s- ’ [39] and k, can be neglected in eqn. (8) (k,> l@ s- ‘). So, the triplet half-life is determined by the first two terms on the right-hand side of eqn. (9). k, is 6700 s-’ [39], kTC is equal to 3.1 x lo8 dm3 mall’ s-’ [16] (1.3 X lo9 dm3 mol-’ s-’ in 0.6 mol NaCl dmP3, determined as described in ref. 16) and krr is equal to 1.5 X lo9 dm3 mol-’ s-’ [16]. The micelle concentration [M] can be calculated by use of the classical relationship [M] = ([NaTC] - CMC) /N

(IO)

where N denotes the mean aggregation number. In the presence of 0.6 mol NaCl dmw3, the distribution of the aggregation number of NaTC

53

micelles is relatively sharp, the mean aggregation number N= 15 [42] and the CMC value is equal to 3 mmol dme3 (vide infra). Without NaCl, the polydispersity of the NaTC micelles is high and micelles with very different aggregation numbers have been detected [25, 421. Under these conditions, the relationship (10) may only lead to an approximation of micelle concentration, by use of CMC= 3 mmol dmm3 (wide infra) and N= 4 [30]. In this paragraph, values given in parentheses refer to solutions containing 0.6 mol NaCl dmm3. Above 15 (20) mmol NaTC dmh3, the triplet half-life is constant for [Fe(CN):-] G 5 x 10h5 (2 X lo-‘) mol dmM3 (Fig. 4). Moreover, at these NaTC concentrations, we have already concluded that triplet-triplet annihilation is negligible. Under these conditions, we obtain k,[M] + kT> i.e. k,[M] + 6.7 x lo3 > 3.1 x lo8 x k,C[WCJ963-l, 5x10-’ (1.3~10~~2~10-~) or k,[M]>1.5x104 (2.5 x 10”) s - ‘. The relationship (10) and the data recalled above allow the calculation of micelle concentration: [M] = 3 (1) mm01 dm-3 at 15 (20) mmol NaTC dm-3. Finally, k, > 5 x lo6 (2.5 x 10’) dm3 mol - ’ s- ‘. The screening effect of sodium ions could explain the easier access for RB triplet state to NaTC micelles in the presence of NaCl. The lower limits of k, and k, obtained for NaTC aggregates compare well with the values found for NaDS spherical micelles for which k, is equal to 3.7 x 10’ dm3 mol-’ s-l and k, ranges from 1.5 x 10s to 17.5 X lo5 s-l depending on NaDS concentration

WI3.3. The semioxidized radical 3.3.1. Transient spectrum

When an air-saturated aqueous solution of RB and NaTC is excited by a flash, the transient difference spectrum observed exhibits a broad absorption band with maximum at 470 nm (Fig. 6) which looks like the spectrum of semioxidized RB recorded in other air-saturated media [15]. Similar spectra are recorded in the presence of lo-* mol NaOH dmw3 and 0.6 mol NaCl dmP3. The study of semioxidized RB in unbuffered water is facilitated by the absence of any protonation of this radical in the pH O-14 range [43]. The decay of semioxidized radical has been followed at 465 nm. 3.3.2. Decay in air-saturated solutions The semioxidized radical is more conveniently studied in air-saturated solutions [15]. The halflife of semioxidized RB has been measured at different NaTC concentrations in unbuffered, lo-’ mol NaOH dme3, lo-* mol NaOH dme3 plus 0.6

A. Seret, A. Vun de Vorst / Rose bengal in aqueous Na taurocholate I

L

470

L30

5X

hlnml

Fig. 6. Difference spectra recorded at different times after the flash excitation of an air-saturated aqueous solution containing 5 pmol RB dm-’ and 5 mm01 NaTC dm-‘.

C

-4

1.f

1.2

L; -1

_i

[NaTC]lmmol

dmm3)

.l

_C

[Na TC] (mmol dmm3) Fig. 7. (a) Evolution of the half-life of semioxidized RB as a function of NaTC concentration. The solutions contain 4.7 pmol RB dm-” and any electrolyte (curve I), lo-* mol NaOH dm-” (curve II), and lo-* mol NaOH dmW3 plus 0.6 mol NaCl dm-’ (curve III) and are equilibrated with air. (b) Dependence of the initial slope of semioxidized RB decay kinetics on NaTC concentration in the same solutions. The initial slope was not always measurable (see text for further details).

mol NaCl dmp3 aqueous solutions. The results of the experiments are plotted in Fig. 7(a). The initial slope k0 of the logarithm of the transient absor-

bance at 465 nm vs. time (decay kinetics of semioxidized RB) has been measured as described in ref. 44 (Fig. 7(b)). I n unbuffered water, k. is always measurable. However, in the electrolytic solutions, the half-life of semioxidized RB decreases rapidly and reaches virtually the resolution limit of our flash photolysis equipment. Under these circumstances, the measurement of the initial rate is not possible. In the region where the halflife of semioxidized RB decreases drastically with the increase in NaTC concentration, k,, is a linear function of NaTC concentration (Fig. 7(b)). We have also investigated the quenching reaction of semioxidized RB by Fe(CN)64- in aqueous solutions containing various NaTC concentrations. Plots of k,, vs. Fe(CN)64- concentration are linear and the corresponding quenching rate constant k,, can therefore [15] be determined by linear regression. The values obtained (e.g. k,=(3.9+0.8)x108 dm3 mol-’ s-’ at 6 mm01 NaTC dm-3) are always close to that (kx4= (3.4 kO.7) x 10’ dm3 mol -i s-l) measured previously in neat water [15]. These observations indicate that, in NaTC solutions, the semioxidized radicals which are readily detected are those moving freely in the aqueous phase. Therefore, the plots presented in Fig. 7 can be explained as follows. The semioxidized RB is reactive with NaTC and the radical half-life decreases with NaTC concentration. Above 3-4 mmol NaTC dmm3, the reaction rate between semioxidized RB and taurocholate anion is clearly slowed down. The formation of NaTC micelles can account for this observation, since in these aggregates the reaction site of taurocholate anion would become much less accessible to the radical. Finally, below 4 mmol NaTC dmp3, the linearity of k,, vs. NaTC concentration (Fig. 7(b)) indicates that most of the taurocholate anions are monomeric. The following values for the reaction rate constant between semioxidized RB and taurocholate anion are caldm3 mol-’ s-’ in culated: k,,, = (2.9f0.6)x105 neat water and kx,= (3.7 f 0.8) X lo6 dm3 mol -’ S -r in 10e2 mol NaOH dme3 aqueous solution. In their study of the reaction between hydrated electron and NaTC, Chen et al. [24] found plots very similar to those presented in Fig. 7(b), the break occurring around 5 mmol dme3. They proposed an interpretation identical to that presented above. The larger value of kXTC in electrolytic solutions can be easily understood on the basis of the influence of ionic strength on reactions between ionic species [45, 461, notwithstanding a possible pH influence. Indeed, for semioxidized eosin Y, it has been shown that the higher is the

A. Seret, A.

pH, the larger is the rate constant with anionic compounds [44].

Van de Vorst / Rose bengal in aqueous

for reaction

3.3.3. N2-saturated solutions Because of the existence of the reaction between semioxidized RB and taurocholate anion and the well-known recombination reaction between semireduced and semioxidized RB radicals [19], the half-life of semioxidized RB in basic N,-saturated solutions falls seemingly below the time resolution limit of our flash photolysis equipment. Consequently, in those solutions, the signal recorded in the 465-480 nm region does not arise from the semioxidized radical but from the triplet state whose absorption spectrum covers the entire visible spectral region [40, 411. This explains why this signal does not show any dependence of its initial height on NaTC concentration and has a half-life identical to that of the triplet state.

4. Concluding

remarks

The RB singlet states are located in the NaTC aggregates. In contrast, the RB first triplet state exchanges between the micelles and the bulk phase. The same situation prevails in NaDS micellar solutions [9, 161. However, the difference in solubility between the singlet states and the first triplet state remains an unresolved problem. It is also interesting to summarize the features of the aggregation process of NaTC which have been highlighted in this work. The NaTC aggregates in which RB can be solubilized exist above 1 mm01 dmm3, well below the CMC region of NaTC [23, 29, 351. However, most of the taurocholate anions are monomeric until 3-4 mm01 dme3 is reached. Above this concentration, the aggregation appears to be extensive and a large number of taurocholate anions are embedded in the aggregates. Moreover, the larger the concentration in NaTC or NaCl, the more pronounced is the hydrophobic character of NaTC micelles. Hence, the increase in NaTC concentration is followed not only by an increase in micelle concentration, but also by a change in micelle structure. All these considerations agree well with more recent studies on the NaTC aggregation process. Kratohvil et aE. [30] have been able to detect the first NaTC aggregates at 0.4 mmol dmP3 and have claimed that the aggregation of NaTC is a very progressive process. The appearance of NaTC aggregates and the subsequent incorporation of some dyes has been reported to start at 3-5 mmol dme3 [25, 29, 351. This is the reason why 3 mmol dmP3 has been chosen here

55

Na taurocholate

as the CMC value in the calculation of micelle concentrations. Several researchers [25,27,28, 30, 36, 421 have noted that the aggregation of NaTC is more pronounced at higher concentrations or in the presence of a sufficient NaCl concentration (0.6 mol dmB3 has been generally used). Moreover, it has been shown that this more pronounced aggregation leads to micelles having a larger hydrophobic character [27, 291. Finally we would like to highlight two observations made during the present work which would be relevant for photobiologists. First, the solubilization sites of singlet and triplet states can be different and migration of triplet states can occur during their short aerobic lifetime. Second, some dye excited species react with the amphiphile molecule. This probably mimics the in vivo situation where reactions between excited dye and some membrane components would be expected. Consequently, at least in the case of RB, NaTC appears to be a mimetic system closer to the biological reality than are synthetic surfactants such as NaDS.

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