Fluorescence in microemulsions and reversed micelles

Analytica Chimica Acta, 208 (1988) 1-19 Elsevier Science Publishers B.V., Amsterdam -

Printed in The Netherlands

FLUORESCENCE IN MICROEMULSIONS MICELLES A Review and New Results

AND REVERSED

G. RAMIS RAMOS”, M.C. GARCIA ALVAREZ-COQUE”, ALAIN BERTHODb and J.D. WINEFORDNER* Department of Chemistry, University of Florida, Gainesville, FL 32611 (U.S.A.) (Received 4th August 1987)

SUMMARY The use of fluorescence to study physicochemical structures of alcohol/surfactant/water systems, microemulsions and reversed micelles is reviewed, and the application of these media in analytical fluorescence spectroscopy is discussed. The sodium dodecylsulfate/l-pentanol/heptane/water system is studied by using pseudo-ternary diagrams. Wide areas of existence of thermodynamically stable and optically clear phases (Winsor IV and two liquid crystals) were found both in the absence and presence of sodium sulfate (0.2 M ) . The influence of the composition of media on the fluorescence characteristics of pyrene, benzo [elpyrene, 2-naphthol and p-aminobenzoic acid is studied.

Aqueous micellar solutions, microemulsions, reversed micelles and liquid crystals are thermodynamically stable organized media which form spontaneously when appropriate amounts of a surfactant and a polar and/or a nonpolar solvent are mixed. The addition of a cosurfactant, usually an alcohol or an amine, can also be necessary. The structure and properties of these media have been described [l-7] and are still the subject of current investigation. Aqueous micellar solutions are formed by surfactants above their critical micellar concentration (CMC ). The surfactant is organized in micelles with diameters of the order of 4-8 nm, by gathering together the hydrophobic nonpolar tails of the surfactant molecules. The hydrophobic region is separated from the aqueous phase by a layer containing the surfactant polar heads and a “shell” of strongly retained water molecules. This layer is often called the “interphase” and, in the case of ionic surfactants, is also called the Stern layer. The properties of the interphase, including rigidity or flexibility, penetrability by water, local ionic strength and pH, are altered by the addition of cosurfac“On leave from Department of Analytical Chemistry, University of Valencia, 46100 Burjasot, Spain. bOn leave from Laboratoire des Sciences Analytiques, Universiti Claude Bernard, Lyon 1,69622 Villeurbanne Cedex, France.

0003-2670/88/$03.50

0 1988 Elsevier Science Publishers B.V.

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tants and also by salts in the case of ionic surfactants. Surfactant/alcohol /water and surfactant/alcohol/brine systems have been particularly investigated. Microemulsions are formed by further addition of a nonpolar solvent which is dissolved in the interior of the surfactant aggregates. As the content of nonpolar solvent or “oil” increases, the water can lose its role as the continuous phase (Ll structure), and a semicontinuous structure is formed. Eventually, at higher contents of oil, a reversed micellar system can be found as a dispersed aqueous phase in a continuous nonpolar solvent (L2 structure). Some surfactants, e.g., Aerosol-OT [ AOT, disodium bis (2-ethylhexyl)sulfosuccinate] , form reversed micelles even in the absence of cosurfactant. When a semicontinuous structure spontaneously organizes in alternate sheets of polar and nonpolar regions, separated by parallel surfactant layers, the medium is called a liquid crystal. Applications are found in such diverse fields as enhanced oil recovery [1,2,8-lo], simulation of biological membranes [ 11-131, catalysis [ 14,15], cleaning industries [ 161 and photochemical energy storage [ 17,181. The analytical applications of aqueous micellar solutions have been reviewed [ 191. Organized media offer a wide variety of possibilities for the modification of many solvent properties. Particularly useful are their capabilities to co-solubilize polar and nonpolar substances and modify reaction rates and reaction pathways. By using a micellar solution, liquid-liquid extraction can be avoided, which has permitted the development of faster and simpler analytical procedures [ 19,201. The sensitivity of molecular absorption and luminescence measurements can also be enhanced [2,6,19,21]. Besides, micellar solutions have provided a convenient way to observe liquid-phase phosphorescence at room temperature [ 22-241. Microemulsions and reversed micelles offer more possibilities than simple aqueous micelles for the development of new analytical procedures. However, only a few applications have been developed so far. Micro-emulsions and reversed micelles are optically clear, isotropic systems because of the small size (usually 6-60 nm) and stochastic disorder of the dispersed phase droplets, therefore being useful for molecular spectrometry. However, micro-emulsions exhibit to some extent the Tyndall effect with visible light. Liquid crystals are also transparent media, although they are not isotropic. Because of their industrial and commercial importance, many efforts have been devoted to the physicochemical study of microemulsions and reversed micellar systems. Fluorescence, together with other spectroscopic and electroanalytical techniques have been used. In this paper, the literature related to the use of fluorescence in these media is reviewed and their potential applications in analytical fluorescence spectroscopy are discussed. For demonstrative and comparative purposes, some new data are also given. The sodium dodecylsulfate (SDS ) /l-pentanol/heptane/water system is studied by using pseudo-ternary diagrams. This system presents a wide range of existence of thermodynamically stable and optically clear phases which are not reduced in

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the presence of salt (sodium sulfate), even at an ionic strength of 0.2 M. Two extensive areas of liquid crystals are also formed. The influence of the medium composition on the fluorescence spectral characteristics of some polar and nonpolar model compounds is also studied. EXPERIMENTAL

Heptane and hexane (high-purity solvents, Burdick and Jackson, Muskegon, MI) were found to contain no detectable fluorescence impurities. The SDS (99% pure, Sigma Chemical Co.) exhibited an acceptably low fluorescence background after being washed ten times with hexane in a solvent filtration device provided with nylon-66 filters of 0.45pm pore diameter (Rainin Instruments Co., Woburn, MA). 1-Pentanol (Aldrich Chemical Co.) was distilled and only the low fluorescent fractions were used. Water was of nanopure grade (Barnstead Sybron Corp., Boston, MA). Sodium sulfate decahydrate (Fisher Scientific Co.), p-aminobenzoic acid and 2-naphthol (Eastman Kodak Co. ) , pyrene and benzo [ e ] pyrene (Aldrich Chemical Co. ) were used as received. Pyrene and benzo [ elpyrene stock solutions were prepared in heptane. pAminobenzoic acid and 2-naphthol stock solutions were prepared in water. The different SDS/l-pentanol/heptane/water stock mixtures were prepared by weighing the components. Aliquots (3.00 ml) of each medium were pipetted into dry l-cm standard cuvettes, and the fluorophores were added with 5-~1 Hamilton syringes. The cuvettes closed with teflon caps were shaken until a stable fluorescence signal was observed. All measurements were corrected for the fluorescence of the blank. No attempt was made to eliminate oxygen. A Perkin-Elmer LS-5 luminescence spectrometer provided with a model 3600 data-base station was used. Excitation and emission slits were both set at 3 nm, and a 305nm cutoff filter was used in the filter holder on the emission side. REPRESENTATION OF TERNARY AND QUATERNARY SYSTEMS

A ternary or pseudo-ternary diagram is a useful tool for the representation and study of microemulsions and reversed micelles, because these media are composed of three or four substances. Usually, the lower left corner of the diagram is assigned to the mass proportion of the water or salt solution, the lower right corner to the nonpolar solvent and the upper corner to the surfactant. When a co-surfactant is also added, the diagram is constructed for a fixed co-surfactant/surfactant ratio, and the upper corner represents the mass percentage of the mixture of these two components which is called the active blend. The diagram can show different regions corresponding to the formation of several monophases or mixtures of separated phases. Five regions are recognized in the classification of Winsor [ 251. These regions are a liquid crystal

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(an oil phase in equilibrium with a microemulsion, Winsor I), an aqueous phase in equilibrium with a microemulsion (Winsor II), a three-phase system (oil phase, aqueous phase and microemulsion, Winsor III) and a micro-emulsion (Winsor IV). Freshly prepared or after stirring, the Winsor I, II and III regions have.the appearance of milky emulsions and slowly separate into two or three phases. Microemulsions and liquid crystals are clear and stable systems and can be distinguished from the other multiphase regions by simple visual examination. The liquid crystals can easily be recognized by using two crossed polarizer filters. Polarized light is not completely depolarized by a liquid crystal, but there are different polarization axes within short distances. The extent of the particular orientations depend on the sizes of the parallel lamellar structures, which can be of the order of hundreds of micrometers, or even several millimeters. Therefore, a particular pattern of colors and intensities can be seen through a liquid crystal sandwiched between two crossed polarizer filters and illuminated with white light. The diagrams corresponding to the SDS/l-pentanol/heptane/water system for a 1:2 (w/w) SDS/l-pentanol ratio, in the absence and presence of 20 g 1-l Na2S04*10H,0 are shown in Fig. 1. The different regions on the diagram have been marked according to the classification of Winsor. Points 1-12 on Fig. 1A correspond to the media used for the experiments below. When an inorganic salt is added to an organized medium, important changes can occur, from an initial expansion and shift in the position of the major regions of the transparent monophase media (Winsor IV and liquid crystal) to a rapid reduction when more salt is added. The increase in ionic strength is the main cause of these changes, the nature of the salt being of secondary

NaiSQ lOti& (20911)

Fig. 1. Pseudo-ternary diagrams of the SDS/l-pentanol/heptane/water system for a 1:2 (w/w) SDS/l-pentanol ratio: (A) no salt added; (B) 20 g 1-l NazSOI*lOH,O as aqueous phase. See text for the numbers inside the diagrams. LC, liquid crystal.

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importance. Ionic strength and co-surfactant play similar roles, by decreasing the repulsion between the surfactant polar heads and modifying the rigidity of the interphase. Changes in the form of the aggregates from spherical to rodlike structures at high salt concentrations can also be expected [ 26-281. The Winsor IV and liquid-crystal regions are very extensive in this system, and the addition of an inorganic salt can be tolerated almost without reduction to their area, at least up to an ionic strength of 0.2 M. The positions of the regions change only slightly between parts A and B of Fig. 1. SOLUBILIZATION AND CATALYTIC PROPERTIES OF ORGANIZED MEDIA

Solubilization in organized media offers two important characteristics: the high solubility of surfactant-like molecules and the possibility of co-solubilization of hydrophobic and hydrophilic compounds. Although molecules with both hydrophobic and polar groups can be only slightly soluble in either polar or nonpolar solvents, their solubilities in organized media can be considerably higher than in any unstructured solvent. The surfactant-like molecules align with the molecules of the surfactant in the micellar membrane, its hydrophilic and hydrophobic moieties facing their corresponding environments. For example, the solubility of chlorophyll in a sodium cetyl sulfate (SCS) /l-pentanol/mineral oil/water microemulsion is over an order of magnitude greater than in any of the components of the mixture [ 291; similar behavior has been reported for several cyanine dyes in this same medium and in cetyltrimethylammonium bromide (CTAB)/l-butanol/hexadecane/water microemulsions [ 30,311. Co-solubilization of compounds of very different hydrophobic and hydrophilic character, varying from hydrocarbons to inorganic ions, allows the development of chemical reactions which otherwise would proceed only with difficulty. Examples are the formation of complexes between metal ions and tetraphenylporphine [ 32,331, the chlorophyll-sensitized photoreduction of methyl red by ascorbate [ 311, and the formation of o-phthalaldehyde adducts with water-insoluble amines of high molecular weight [ 341. The reagents can interact through a very large interphase, of the order of 100-1000 m2 g-’ of mixture. Furthermore, experimental studies have illustrated changes in reaction rates which in many cases amount to more than three orders of magnitude and, to some extent, reaction pathways can also be modified [ 1,2,12,14,19,35,36]. In this way, side-reactions can be reduced and acid-base and redox equilibria shifted. The possibility of developing new kinetic methods of analysis is particularly promising. Positive and negative catalysis can be due, at least in part, to an increase in the local concentration of reactants or products which are crowded together inside the dispersed phase or in the interphase [ 37,381. However, this mechanism cannot always explain the observed catalytic effects.

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Pronounced gradients of several physicochemical parameters such as polarity, viscosity, electrical field and ion concentrations found in the interphase can influence the transition state of the reaction, e.g., by stabilizing an otherwise transient species [ 331. Orientation effects [ 391 and specific interactions with the surfactant polar heads such as dipole/dipole attraction or hydrogen bonding can also occur. The use of microemulsions in addition to micellar solutions permits the achievement of a higher degree of control on the reactivity and reaction rates than simple unstructured solvents. This can be mainly accomplished by modifying the properties of the interphase and the size of the aggregates. As fluorescence studies have shown and as discussed below, both parameters can be “adjusted” to the particular requirements of the reaction under study. For the reactions investigated in reversed micelles, it has often been observed that a decrease in the reaction rates occurred as water is added. Indeed, at low water contents, substrates located in the v.icinity of the interphase interact with the polar heads of the surfactant. These interactions can be quite strong and selective, but are modified by addition of water, because the surfactant head groups, acting as catalysts, are deactivated by hydration [ 40-421. As water is added, redistribution of the substrates between the hydrated shell of the micellar membrane and the free-water pool, together with the evolution of the shell itself, account for the changes in reactivity in the medium. Microemulsions and reversed micelles offer some additional advantages over the use of simple micellar solutions. The capacity of the solvent as reagent reservoir can be adjusted to the requirements of the experiment by modifying the proportions of polar and nonpolar components. In many microemulsions and reversed micellar systems, the mass fraction of a solvent can be varied over a fairly wide range (e.g., O-80% ) without destruction of the monophase system. Besides, microemulsions are far better vehicles than micelles for solubilizing hydrophobic molecules, such as chlorophyll [ 29,371 or tetraphenylporphine [ 321, which are too big to be easily accommodated in a micelle [ 381. Organized media can also offer a high degree of protection against photodecomposition. In a study with merocyanine-540, Dixit and Mackay [ 301 observed complete bleaching of a solution of the dye in water and in 1-pentanol in less than 5 min and a loss of about 20% in ethanol and SDS micellar solutions over a period of 70 min. In contrast, there was no detectable degradation of the dye in SCS and CTAB microemulsions. Enhanced photostability is probably related to a lower photo-ionization yield. Gregoritch and Thomas [ 381 observed a photo-ionization yield of about 38% for pyrene in a potassium oleate/hexanol/hexadecane/water microemulsion with respect to the photo-ionization yield found in SDS and potassium oleate micellar solutions. The larger distance between the pyrene molecules and the interphase in the microemulsion structure ( 2 5 nm) with respect to micelles decreases the efficiency of escape of the photo-produced ion-pair and makes

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recombination easier. Enhanced protection was not observed for pyrenebutyric acid, which resides in the interphase. The particular properties of reversed micelles as solvent media have been applied by Memon and Worsfold to the determination of lipase with Li-methylumbelliferyl heptanoate [ 43 ] and to the determination of water-insoluble primary amines ( C6-C10) with o-phthalaldehyde [ 34,431. In both cases, a flowinjection system with a fluorimetric detector was used. Lipase can be determined in an aqueous environment by using 2-methoxyethanol to solubilize the substrate. However, several analytical characteristics of the method can be substantially improved if the reaction is done in a dioctyl sulfosuccinate (DOSS) /heptane reversed micellar system. Because heptane is the major solvent, higher amounts of substrate can be dissolved and addition of 2-methoxyethanol is not necessary. The enzyme is solubilized in the polar surfactant pools. The higher solubility of the substrate, together with some protection against nonenzymatic hydrolysis, lead to a five-fold extension in the linear dynamic range. The modified procedure for the determination of primary amines takes place also in a dioctyl sulfosuccinate/heptane system. The amines are dissolved in the heptane continuous phase and an aqueous alcoholic solution of o-phthalaldehyde and 2-mercaptoethanol is dispersed into the polar pools. An inverse relationship was found between the chain length of the amine and the sensitivity. The effect was attributed to the increased penetration into the polar droplets by the amines with shorter chains. Analytical applications based on the properties of liquid crystals do not appear to have been reported. Two liquid crystals of very different composition were observed in the system studied here, one of them having a high water content and the other having a high oil content. At the molecular level, viscosity and polarity changes are small when a microemulsion is transformed into a liquid crystal by a change in composition, with only the external macroscopic viscosity increasing significantly. Macroscopic viscosity is related to the size of the lamellar structures, and decreases when the liquid is stirred (thixotropic properties). This allows the elimination of entrapped air bubbles by sonication. The gel-like properties of an unstirred liquid crystal can be of analytical interest. FLUORESCENT PROBES IN AQUEOUS CONTINUOUS-PHASE

SYSTEMS

Fluorescent probes have become widely used for the study of the structure of organized media, as well as for biochemical studies involving biological membranes, proteins and other macromolecules. Fluorescent probes for organized media are compounds which solubilize preferentially at the micellar membrane. Peak position, fluorescence quantum yield, fluorescence polarization, excimer formation, and/or the ratio between the intensity of different

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vibronic bands of the fluorescent probe are sensitive to the changes in local polarity and/or viscosity. With fluorescent probes, the critical micelle concentration [44,45], size of the aggregates [46], and properties of the micellar membrane 147-511 can be established. Pyrene and its derivatives are the most popular fluorescent probes for sensing local polarity in organized media when the continuous phase is water. Particularly, the ratio of the intensities of the first (ca. 372 nm) and third (ca. 383 nm) vibronic bands of pyrene, 1J13, is a very sensitive parameter [28,45,48,50,51]. The pyrene I,/& ratio decreases, varying from 1.87 in water to ca. 0.60 in pure aliphatic hydrocarbons. Intermediate values are obtained in organized media. Some values, obtained by the authors, corresponding to SDS/l-pentanol/water and SDS/l-pentanol/heptane/water systems, are shown in Table 1. In micellar solutions, pyrene is located in the interphase and, probably, is displaced towards the nonpolar interior when a co-surfactant is added [28,37,38,52-541. Thus, as can be seen in Table 1, when less than 4% (w/w) pentanol was added to a 5% (w/w) SDS aqueous solution, the pyrene I& ratio decreased from 1.00 to 0.83. When more pentanol was added (from 4% to lo%)), no further changes in polarity were sensed by the probe, indicating some kind of saturation of the pentanol in the micellar membrane and probably the formation of swollen micelles, with an alcohol-containing core. In the quaternary system, two series of experiments were done: points l-7 (Fig. 1A) correspond to systems with the same heptane/water ratio, but with increasing amounts of active blend, points 7-12 correspond to systems with the same active blend/water ratio, but increasing amounts of heptane. Points TABLE 1 Values of the pyrene Z1/Zaparameter SDS/l-pentanol/water Amount of lpentanol (% ) 0

2.6 4.2 4.7 5.1 5.9 8.2 10.0 “5% SDS in all cases.

systems”

SDS/l-pentanol/heptane/water

systems

ZJZ3

Point no. (see Fig. 1)

Z1lZ3

1.00 0.95 0.83 0.83 0.82 0.83 0.83 0.84

1 3 5 7 8 9 10 12 Pure heptane

0.84 0.85 0.88 0.88 0.88 0.78 0.70 0.64 0.58

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1 and 2 correspond to Ll structures, whereas points 5-7 are probably bicontinuous structures, because of the high content of pentanol and their location beyond the liquid crystal and Winsor II regions. As shown in Table 1, the polarity sensed by pyrene in points l-7 was approximately constant, suggesting that pyrene is in an environment with an almost constant alcohol/surfactant ratio. When heptane was added to system 7, the polarity sensed by pyrene decreased as is deduced from the change in the value of the I,/& ratio, from 0.88 to 0.64 (Table 1) . These values seem to indicate that points 7 and 8 are still bicontinuous structures, which evolve to L2 as more heptane is added. Heptane dissolves in the alcohol-rich microdomains which become larger and less polar. At the same time, pyrene distribution in these environments is favoured. If the 1J& values for point 12 (0.64), and for pure heptane (0.58) are compared, it can be concluded that the solubilization site of pyrene in L2 structures is not totally nonpolar. Pyrene moves freely in the interior of the nonpolar microdomains, and its emission spectrum depends on the average polarity of the different microenvironments sampled by the excited state during its long lifetime. This explains the relatively high value of the parameter I,/&,, even when the proportion of nonpolar solvent is large [37,51]. Another factor that must be considered is that micellar solutions, surfactant/alcohol/water systems, and microemulsions differ in the compactness of the micellar membrane, surface charge density, and extent of water penetration. For example, surfactants with smaller polar head groups, such as SDS compared to CTAB or Brij-35, are less penetrated by water. Addition of small amounts of an alcohol causes also an exclusion of water and a tightening of the micellar membrane. Changes in the relative amounts of the components also lead to changes in the size of the microdroplets which induce a different average distance between the pyrene molecules and the interphase. The pyrene 1J& parameter senses all these factors indiscriminately [ 451. Polar-tailed derivatives of pyrene, such as pyrenebutyric acid, pyrenesulfonic acid and pyrene-3_carboxaldehyde, are permanently retained at the interphase and spectral changes are less affected by the size of the aggregates. Local viscosity measurements can be done by fluorescence depolarization [ 55-581 and excimer-formation studies [ 28,38,48,59,60]. Excimer formation decreases when solvent viscosity increases, which is reflected by the excimerto-monomer peak intensity ratio, 1,/I,. Dipyrenylpropane, which easily forms an intramolecular excimer, is a better probe than pyrene for microviscosity measurements. Dipyrenylpropane excimer fluorescence can be observed at a very low concentration, and does not depend on as many factors as pyrene excimer formation [28,47,61-631. The microviscosity sensed by a probe in a particular environment can be very different from the external or global viscosity of the medium [ 541. Molecules attached to the aggregates can also show dramatic changes in

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B

~.~.L._._

. d

-.-.--.---.-. 5

I

0.5 ,-P.nt,nc.l,*D*

2

15 ,*‘L

1

20

r.tlo,

40

60

HEPTANE

60

IC

(W% )

C

20

a

30

40

50

60

m

I

ACTIVE BLEND (w%)

Fig. 2. Influence of various system parameters on the fluorescence emission intensity of some compounds: (A) effect of the 1-pentanol/SDS ratio (with 5% w/w SDS); (B) effect of the heptane content; (C ) effect of the active blend content. Compounds: ( 0 ) pyrene; (W ) 2-naphthol; ( 0 ) benzo [e ] pyrene; ( 0 ) p-aminobenzoic acid. (See Table 2 and Fig. 1A. ) TABLE 2 Experimental

conditions

Compound

Pyrene Benzo [e ]pyrene p-Aminobenxoic acid P-Naphthol

used in Fig. 2 Concentration (ng ml-‘)

Excitation wavelength

53 58 110 123

275 290 269 275

(nm)

Emission wavelength

(nm )

393 388 340 354

fluorescence quantum yield caused by local polarity and viscosity changes. For example, the emission of pyrene-3-carboxaldehyde in water, SDS/l-pentanol and pentanol/dodecane mixtures, and SDS/l-pentanol/dodecane/water microemulsions is at least ten-fold more intense than in pure dodecane [ 371. The

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quantum yield of merocyanine-540 is twelve-fold higher in CTAB micelles than in water [ 301. Fluorescence intensity variations over two orders of magnitude have been reported for the probe 4-heptadecylumbelliferrone in petroleum sulfonate TRS lo-410/alkyl alcohol/alkane/salt solution systems upon variation in the salt content and of the alcohol chain length [ 641. The effect of micellar solutions of SDS, cetyltrimethylammonium chloride (CTAC) and Triton X-100 on the fluorimetric determination of pyrene was investigated by Singh and Hinze [ 211. A 3-16-fold increase in sensitivity and a more extended linear dynamic range, compared to the use of ethanol, were observed. In Fig. 2, the variations in maximum emission intensity of several fluorophores in SDS/l-pentanol systems and SDS/l-pentanol/heptane/water microemulsions obtained by increasing the contents of 1-pentanol, heptane and active blend are presented. The corresponding experimental conditions are summarized in Table 2. The fluorescence quantum yields of the nonpolar fluorophores decrease continuously from micellar solutions to alcohol/water mixed systems and to oil-rich microemulsions. Pyrene, which is located very close to the interphase, shows the largest intensity variations. The highest intensity was obtained in pure micellar solutions (5% SDS) and the lowest in heptanerich media, the ratio of intensities being about 34. Benzo[e]pyrene, which is probably permanently located in a more hydrophobic environment than pyrene and, therefore, at a somewhat larger distance from the interphase shows a smaller influence of the medium composition. 2-Naphthol andp-amino-benzoic acid are probably associated with the aqueous phase and show very small intensity changes. Furthermore, the smooth shapes of the curves in Fig. 2B indicate a continuous change in the physicochemical structure of the aggregates, without sharp transitions, from a bicontinuous structure (point 7) to an L2 structure (point 12). In this last medium, the fluorescence intensities of pyrene and benzo [e ]pyrene are similar to the intensities in pure heptane. Red shifts in the emission spectrum up to 30 nm can also be expected for a variety of compounds upon increase in local polarity, as has been observed for pyrene-3-carboxaldehyde, dihexylaniline, N-phenylnaphthylamine and dimethylaniline [38,65]. The position of the maximum helps to establish the solubilization site of the probe. For the compounds and systems investigated in this work, no blue or red shifts larger than 2 nm were found. FLUORESCENT PROBES FOR REVERSED MICELLAR SOLUTIONS

For the study of reversed micelles, in which the continuous phase is hydrophobic, pyrene is not an appropriate probe. In this case, anilinonaphthalene derivatives and, particularly, 1-anilino-8-naphthalene sulfonic acid (ANS) are frequently used. These compounds are very important fluorescent probes for the study of proteins and other biological materials [ 581. The ANS molecules

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prefer polar environments and the emission peak shifts to longer wavelengths as the polarity increases, which is often, but not always, accompanied by a decrease in the fluorescence quantum yield. Besides, ANS does not fluoresce in pure hydrocarbon solvents [ 561. Therefore, ANS is a suitable probe for sensing polarity in hydrophobic environments, such as reversed micelles. Wong et al. [56] used ANS for the study of DOSS/heptane/water and DOSS/dodecane/water reversed micellar systems. In water-free DOSS/alkane solutions, ANS displays a very intense fluorescence, which appears as a broad structureless band with a maximum at 450 nm. This fluorescence is drastically quenched by the addition of water, with a concomitant red shift of the maximum. The changes of the parameters were most significant in a region where the water content was relatively small (O-2% ) and approached a plateau at larger radii of the water pools. However, the effective polarity sensed by ANS in a water pool with a radius as large as 7 nm is still smaller than the polarity in pure water, as can be deduced from the comparison of the fluorescence quantum yields and position of the emission maxima in both media, from 0.018 and 484 nm to 0.0038 and 510 nm, in the larger water cluster and in pure water, respectively. The sharpest changes in these parameters occur precisely at a point where the water content of the micelles falls below the molar ratio [ Hz01 / [ Na+ ] = 6, that is, when the hydration sphere of the sodium ions of the dioctyl sulfosuccinate salt is complete [ 56,661. Similar results were found by Atik and Thomas [ 671 for a potassium oleate/ hexanol/hexadecane/water system using ANS and pyrene-3carboxaldehyde, and by Kumar and Balasubramanian [68] for a Triton XlOO/hexanol/cyclohexane/water system, also with ANS. Many molecules dissolved in reversed micellar systems show emission peak shifts and changes in the fluorescence quantum yield upon small variations in the system composition, particularly at low water contents. A red shift and an increase in the quantum yield upon addition of water have been found for ophthalaldehyde adducts with amines (OPA adducts) in a DOSS/heptane/water system [ 341. The increase in the fluorescence quantum yield reached a maximum at a water/DOSS ratio of about 20. Zinsli [ 571 studied the structure of the DOSS/isooctane/water reversed micellar system, using the fluorescent probes 1-aminonaphthalene-4-sulfonic acid (AMNS) and 2- (N-tetradecyl)aminonaphthalene-6-sulfonic acids (TANS). When dissolved in nonpolar solvents, these compounds show lower quantum yields and the spectra are more structured than in polar solvents. In DOSS/ isooctane/water micelles, AMNS is in a water-like surrounding, and the fluorescence intensity decreases when the content of water increases. In contrast, TANS is quite tightly bound to the micellar membrane, and the fluorescence intensity is much less dependent on the water content.

13 THE USE OF QUENCHERS WITH FLUORESCENT PROBES

Information about the location of a fluorescent probe and penetrability of the micellai membrane can be obtained by using fluorophore/quencher interactions. Quenching techniques can also be used to determine the average size of the microemulsion droplets [ 37,691. For a given fluorophore/quencher pair, the quenching efficiency mainly depends on the location and mobility of the molecules or ions involved. For example, in anionic surfactant/water interphases, pyrene fluorescence is easily quenched by hydrophobic molecules such as iodoheptane [ 37 3 and diiodomethane [38], with more difficulty by cationic quenchers which reside in the interphase, such as Tl +, CL?+ or methylviologen (N,N’-dimethyl-4,4’-bipyridinium, MV2+), and is not affected by iodide which is repelled from the interphase by electrostatic repulsion. However, iodide quenches the fluorescence of anionic fluorophores located at the interphase of anionic micelles, such as pyrenebutyric and pyrenesulfonic acids [ 38,51,53,67]. Some ubiquituous quenchers such as oxygen and dimethylaniline efficiently quench the fluorescence of all these molecules indicating that there is no significant barrier to their movement throughout the system [ 37,67,70,71]. The influence of the mobilities of fluorophore and quencher can be illustrated by the different quenching efficiencies exhibited by hexacyanoferrate (III) and methylviologen on the fluorescence of tris (bipyridyl) reversed ruthenium(II), Ru(bipy)g+ , in a potassium oleate/hexanol/water micellar system [ 671. The quenching efficiency of hexacyanoferrate (III) is much greater than that of methylviologen, as the latter quencher, as well as the fluorophore, are bound to the negatively charged surface of the water pool, while the former is free to move in the pool itself and approaches the excited state of the fluorophore more readily than methylviologen. Similarly, in a DOSS/heptane reversed micellar system with less than 1% of water present, Cu2+ and Tl+ show very little influence on the fluorescence of ANS. The movement of the quencher ions is almost completely prevented under conditions where the water content of the micelles is merely sufficient for solvation of the ionic species present inside the water pools. The quenching efficiency rapidly increases as more water is added [ 561. Handa et al. [ 721 studied the HED/l-hexanol/n-decane/water reversed micellar system [ HED, heptakis (ethyleneglycol) mono-n-dodecyl ether] by using the fluorescent probe [Ru(bipy),]Cl,. For low water content, the fluorescence spectrum of the probe had a maximum at 582 nm, which disappeared on addition of water, giving rise to another maximum at 615 nm. When the water content was about 0.25%, the two maxima were observed concomitantly. The hydrophilic quencher, Fe ( CN)g-, preferentially quenched the peak at 615 nm. The behavior was interpreted as being due to the distribution of the probe between two different environments present in the water pool; the center

14

of the pool, where free water is available, and the hydrated poly (ethyleneglycol) shell of the micellar membrane. The probe enters the hydrated shell probably as an ion-pair, whereas the solubility of the quencher in this less polar environment is limited. Quenching by oxygen Quantitative procedures are more convenient if deaeration is not necessary. Besides, deaeration with a purge gas can be troublesome when foam is likely to be formed. Although many compounds still show intense fluorescence in aerated solutions, the quenching effect of oxygen can be very important. The solubility of oxygen in nonpolar solvents is about one order of magnitude higher than in water and, for this reason, the solubility of oxygen in organized media increases with the mass proportion of the nonpolar components [ 70,711.Furthermore, oxygen is distributed between the different regions of the microstructured medium according to its solubility and, therefore, its concentration is higher in the hydrophobic regions [37,38,56,67,70]. Consequently, oxygen quenches more efficiently fluorophores which are located in a hydrophobic environment, such as pyrene in microemulsions, whereas fluorophores such as pyrenebutyric and pyrenesulfonic acids, which are located in the interphase, experience an oxygen concentration approaching that of water and thus, are quenched to a lesser extent [ 37,381. Similarly, the rate of quenching of pyrene by oxygen in SDS micellar solution, where pyrene is located at the open surface of the micelles, is the same as in pure water [ 371. The variations in the emission intensity observed in Fig. 2 must be partially attributed to quenching by oxygen. FLUORESCENT PROBE STUDIES

The properties of the interphase As has been shown with fluorescent probes, the properties of the micellar membrane are strongly dependent on the nature of the surfactant polar heads and the presence of a co-surfactant. Tricot et al. [ 471 applied 13C-NMR, fluorescence and light-scattering techniques to elucidate the function and site of the co-surfactant molecules within the microemulsion droplet. An SDS/l-pentanol/hexadecane/water system was used. It was found that for an Ll structure (aqueous continuous phase), the fraction of 1-pentanol associated with the interphase was only 20-35%, most of the co-surfactant being buried in the interior of the aggregates. Water penetration into the droplets was found to be insignificant, even in the absence of alkane. Another interesting finding is a variation in the degree of rigidity with the distance from the surface of the aggregate. Maximum rigidity is located between the second and fourth carbon atoms of the alcohol chains. However,

15

the rigidity found in the center of the droplet core is still considerably higher than that of a homogeneous hydrocarbon solvent. Lianos et al. [48] studied the variations in microviscosity sensed by dipyrenylpropa~ne when different alkyl alcohols were added to SDS micelles. Microviscosity decreased very rapidly for the first alcohol additions and then much more slowly, as if some saturation occurred. Local viscosity depends little on the nature of the alcohol but is always smaller in the presence than in the absence of alcohol. Similarly, the polarity sensed by pyrene rapidly decreases as an alcohol is added to a micellar solution. This result has been mainly attributed to the removal of water from the interphase and its replacement by the less polar alcohol molecules whose hydroxyl groups are most likely anchored at the micelle surface [ 481. Russell and Whitten [49] investigated the changes introduced in the SDS micellar membrane when an alkyl alcohol was added. The study was done with highly hydrophobic fluorescent probes (truns-stilbene derivatives) and the highly hydrophilic quencher, methylviologen ( MV2+ ) . The fluorescence intensity of the stilbene derivative, C6H,-CH = CH-C&H,- ( CH2),-COOH, in the absence of quencher, increased with the heptanol concentration, until a maximum was reached at a heptanol/SDS mole ratio of about 2, and then it began to decrease. With MV2+ present, the fluorescence intensity started at a much lower level, because of the formation of a probe/quencher complex, but rapidly increased with the heptanol concentration. The addition of alcohol prevented the interaction of probe and quencher more than can be accounted for by the simple increase in micellar volume, which means that the micellar membrane has become less penetrable, and probe and quencher can no longer interact so easily. The same conclusion was reached by using other stilbene derivatives in SDS/l-pentanol/dodecane/water and SDS/l-pentanol/water systems. The results are consistent with a very open structure for the simple SDS micelle, with very little capacity for separating hydrophobic from hydrophilic regions. However, upon addition of alcohol, the probe becomes more protected. The distance between the surfactant charged heads increases and so, the repulsion between them decreases [ 731. In addition to tightening the interface, the presence of the co-surfactant causes the displacement of the stilbene derivative towards the interior of the aggregate. Zana et al. [ 741 showed that the introduction of polyoxyethylene, a polymer which interacts with SDS, in a SDS/l-pentanol system can produce aggregates with very low aggregation numbers, n (the average number of surfactant molecules per aggregate). In this case, the changes in n and the parameter II/I3 of pyrene were found to be correlated. As n decreased, the micelle-solubilized pyrene sensed an increasingly polar microenvironment indicating that the micelles became more penetrable by water. These studies indicate how the properties of the micellar membrane can be controlled, from loose aggregates with little separation of hydrophobic from

16

hydrophilic regions to well-organized assemblies capable of sequestering nonpolar solutes in a hydrophobic interior, much less accessible to polar solutes. The size of ‘the aggregates Lianos and co-workers [ 28,48,50] studied the changes in the state of aggregation of the surfactant when going from pure micellar solutions to mixed surfactant/alcohol systems and to microemulsions. When 1-pentanol is added to an SDS micellar solution, large variations of n are produced. The co-surfactant is solubilized in the palisade layer of the micelle leading to a decrease in the charge density and, therefore, to an increase in the degree of micelle ionization. As a result, the micelles break down into smaller ones. This effect occurs as long as the micellar concentration is sufficiently low, i.e., when the electrostatic repulsions between micelles are weak. At high surfactant concentrations (small intermicellar distance), or high alcohol concentrations (extensive micelle ionization), however, the electrostatic repulsions between micelles become large. It may then become more favorable for the micelles to grow in size than to be closer and closer, and n increases rapidly. A similar behavior has been observed for the tetradecyltrimethylammonium bromide/pentanol system [ 73,751. When an oil is added to a surfactant/alcohol system, the value of n can increase, remain constant or decrease, depending on the nature, chain length and isomerism of the oil [ 501. For long-chain alkanes (as dodecane is for the SDS/pentanol system), n decreases, goes through a minimum, and then increases as the alkane mass content becomes larger. The initial decrease was attributed to a change of shape of the aggregates from a non-spherical to a spherical shape. At a higher alkane content, the aggregates are more and more swollen with oil, with a resulting increase in n. If an aromatic hydrocarbon, e.g., toluene, is used instead of an alkane, an increase in n is always observed. The difference in behavior has been attributed to the fact that alkanes can only dissolve in the micellar core, whereas aromatic hydrocarbons usually dissolve both in the core and in the interphase layer [ 48,76,77]. Toluene can play the same role as an alcohol, and the addition of small amounts of toluene is equivalent to the addition of more alcohol. As the concentration of toluene increases, an oil core starts to be formed. Conclusions Microemulsions, reversed micelles, and liquid crystals have not been used extensively in analytical chemistry. Advantages compared to the use of simple micellar solutions can be found in the determination of large surfactant-like compounds, as well as in kinetic methods of analysis. Ternary or pseudo-ternary diagrams can be very useful for selecting the adequate medium and for optimization purposes. New possibilities have been opened by better knowledge of the structures of

17

these media provided by physicochemical studies. These studies have shown how the properties of the interphase and the sizes of the aggregates can be modified. In this way, the catalytic properties of the interphase, its permeability to polar or nonpolar compounds, and the separation of reactants and/or products can be controlled. The attachment of a catalytic surfactant-like derivative to the interphase can also be imagined. Studies with fluorescent probes have indicated which changes can be expected in the position and intensity of the fluorescence emission peaks of a particular compound, when the medium composition is modified. These studies also suggest the possibility of achieving selective quenching of some fluorophores in the presence of other fluorescent compounds. In this way, the sensitivity and selectivity of fluorimetric procedures can be improved. G. Ramis Ramos and M.C. Garcia Alvarez-Coque thank the “Conselleria de Cultura, Educacio i Ciencia de la Generalitat Valenciana” for the grants which made possible their stay in Gainesville.

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