Characterization of the Structural Transitions in CTAB Micelles Using

Article pubs.acs.org/JPCC

Characterization of the Structural Transitions in CTAB Micelles Using Fluorescein Isothiocyanate Nor Saadah Mohd Yusof,†,‡ M. Niyaz Khan,‡ and Muthupandian Ashokkumar*,† †

School of Chemistry, Faculty of Science, University of Melbourne, Victoria 3010, Australia Department of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia

‡

ABSTRACT: A fluorescence-based method has been developed to detect the structural changes that occur in micelle systems. The sensitivity of fluorescein isothiocyanate (FITC) has been evaluated for (i) detecting the micellization of cetyltrimethyl ammonium bromide (CTAB) and (ii) probing the concentration dependent aggregation, leading to microstructural changes that occur within CTAB micelles. The critical micelle concentration (cmc) of CTAB has been determined to be 1.35 ± 0.35 mM using the fluorescence spectral characteristics of FITC. Because the experimental conditions have been altered to optimize FITC probing, the cmc is also validated by surface tension and conductivity measurements. To make sure FITC does not affect the properties of micelles, we calculated the micelle binding constant, KM, at different concentrations of FITC using a nonlinear least-squares method. The average KM for [FITC]T ≤ 5 mM is found to be 6575 ± 233. The optical properties of FITC have also been found to be sensitive in response to the changes in the polarity of the microenvironment, caused by the structural changes in CTAB/water system. Two significant observations are noticed from the fluorescence spectra of FITC in CTAB solutions: (i) a decrease followed by an increase in the maximum intensity (Imax) of fluorescence and (ii) a red shift of maximum wavelength (λmax) with increasing concentrations of CTAB. These observations could be correlated with the concentration-dependent microstructural changes in CTAB micelles. On the basis of the experimental observations, FITC is found to be a suitable fluorescent probe for monitoring the changes in CTAB micelle structures.

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INTRODUCTION Micellization is a reversible self-assembly process of surfaceactive materials above a well-defined threshold concentration, known as the critical micelle concentration (cmc).1,2 The morphology of micelles relies heavily on the electrostatic/steric repulsion between the hydrophilic heads and the attractive interactions of the hydrophobic tails. In general, the tail of a surfactant may consist of 8 to 18 carbons.3 The average size of spherical micelles is ∼50 Å with around 40−100 surfactant molecules per micelle unit.4,5 However, manipulation of the electrostatic/steric and the hydrophobic interactions by any means may result in the formation of different structures, such as cylindrical,6,7 wormlike,4,8−10 and vesicles.9,10 Known as the “smart nano-materials” and “living polymers”,11,12 the flexibility of micelles has attracted people from industries as well as the researchers to explore the possibilities and advantages they might offer in various applications including drug delivery. The microstructures of micelles and their changes have been determined by several analytical techniques, such as NMR,13 neutron or X-ray scattering,13,14 chemical probes,15−18 electrochemistry,19 and electrophoresis.20 Micelle structures may be significantly altered by the slightest changes in experimental conditions, such as the addition of hydrophobic counterions. The growth of micelles may be induced by a decrease in the electrostatic repulsion between the micelle heads and an © 2012 American Chemical Society

increase in the hydrophobic interaction between the tails. Such growth may also occur simply due to an increase in the concentration of the surfactants. Because the changes to microstructures during the concentration-dependent growth of micelles may be very subtle, the use of a very sensitive probe is recommended. It is also important to make sure that the probe itself does not significantly affect the process of either micelle aggregation or deformation. This equilibrium has been described in review articles21,22 and textbooks.23,24 Any interaction of the probe with surfactant molecules may result in probe-micelle aggregation, leading to the formation of different type of “impure” micelles, as reported in a system involving cyclodextrin and CTAB.25−27 Fluorescence probing has been a useful method for the characterization of many systems including micelles.28 A search in the literature results in a variety of fluorophore compounds sensitive in detecting the cmc of surfactants. This is mainly associated with the significant changes in the optical properties of the fluorophore due to the presence of surface-active molecules in monomer and micelle forms.29,30 However, a fundamental understanding of the suitability of fluorophores for Received: May 18, 2012 Revised: June 19, 2012 Published: June 22, 2012 15019

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temperature (23.9 ± 1.2 °C). [ ]T is the total concentrations in micelle and aqueous phases. All samples were sonicated by immersing them in a sonication bath (45 kHz, ∼300 W, total volume capacity 20 L) for 5 min to allow sufficient time for FITC to equilibrate in the desired environment. From the literature, it is known that the maximum interaction between FITC and the surfactants could be reached within 3 min of standing time.43 LabCHEM conductivity meter and Analite 2141 surface tension meter were used for conductivity and surface tension experiments, respectively. A negligible change in the temperature during the course of the experiments did not affect the conductivity and surface tension values. UV−visible absorption spectra of the samples were measured using Varian Cary 50 Bio UV−visible spectrophotometer. The fluorescence spectra of the samples were measured using Shimadzu RF5301PC spectrofluorophotometer with the excitation wavelength fixed at 505 nm. Fluorescence spectra were recorded in the emission range 505−700 nm. Fluorescence images were taken using Olympus Panasonic IX71 optical/fluorescence microscope. All measurements were repeated at least twice.

monitoring the interaction between micelles as well as the structural changes that occur above the cmc is still lacking, especially for systems with minimal micelle growth, such as concentration-dependent microstructural changes. In this study, a novel method has been developed to probe the microstructural changes that occur in aqueous CTAB solutions using fluorescein isothiocyanate (FITC) as a fluorescence probe. FITC is a well-known fluorescent label for a variety of systems including proteins, antibodies, and lectins.31−34 It shows sensitive photophysical responses, which depend on its microenvironmental properties, such as polarity,35,36 solution pH,37−39 and H-bonding.40−42 The variations in observable responses include the absorption and fluorescence spectral characteristics and fluorescence quantum yield and lifetime. The interaction of FITC with CTAB is known to follow Langmuir monolayer adsorption with 1:3 ratio due to the negative and positive charges of FITC and CTAB, respectively.43 Furthermore, significant changes in FITC fluorescence spectra were observed with a difference in the polarity of microenvironment surrounding the probe for proteins and different ionic micelle systems.43,44 This provides promising insight into probing the concentration-dependent structural changes that occur in CTAB micelles using FITC. To evaluate the suitability of FITC to determine the micelle properties of CTAB, we measured the cmc of CTAB and compared it with that measured by other conventional methods. We then used FITC for detecting the changes to the structural properties of CTAB micelles.

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RESULTS AND DISCUSSION FITC Spectral Response to Changes in CTAB concentration (Determination of cmc). Prior to evaluating the suitability of FITC for monitoring the concentration dependent microstructural changes in CTAB micelles, the effect of [CTAB] on the emission properties of FITC was investigated. The fluorescent spectra observed in aqueous solutions containing 1 × 10−5 M FITC with varying amounts of CTAB are shown in Figure 1. On the basis of the UV−visible absorption characteristics, an excitation wavelength of 505 nm was used to record the fluorescence spectra. The spectral variations observed in Figure 1 demonstrate the dramatic sensitivity of FITC to the changes in the concentration of CTAB. A gradual decrease in the maximum intensity is observed for CTAB at low concentrations up to 0.3 mM, followed by a significant increase with a further increase in

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MATERIALS AND METHODS Materials. Cetyltrimethyl ammonium bromide (CTAB) and fluorescein isothiocyanate isomer I (FITC) were commercial products of Sigma Aldrich of highest available purity and therefore used as received. There are two possible isomers for FITC: Isomer I with thiocyanate group on the meta position to the carboxyl group of the benzene ring and Isomer II with thiocyanate group on the para position to the carboxyl group of the benzene ring, as shown in Scheme 1. Isomer I has Scheme 1. Structures of FITC Isomers

been used in this study because it can be isolated easily in its pure form and is therefore less expensive than FITC Isomer II. Both isomers are indistinguishable spectrally. The stock solution of FITC was prepared in ethanol. CTAB was prepared in water, and a known volume of FITC in ethanol was added so that the amount of ethanol present is less than 1% in every sample prepared. Methods. Conductivity, surface tension, and fluorescence measurements were carried out with samples consisting of five different concentrations of FITC, [FITC]T = 5 × 10−7, 1 × 10−5, 2 × 10−5, 5 × 10−5, or 7 × 10−5 M, and CTAB in the concentration range, [CTAB]T = 0 to ≤0.02 M at room

Figure 1. Changes in FITC fluorescence emission intensity for [CTAB]T (mM) = 0 (●), 0.005 (○), 0.01 (■), 0.3 (□), 1.2 (◆), 1.5 (◇), and 10 (+) with 1 × 10−5 M FITC. 15020

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the concentration. This transformation is also accompanied by a red shift in the band maximum (from 518 to 529 nm). To explain the results (emission intensity) shown in Figure 1, the existence of various forms of FITC in different solution environments should be considered. Variation in the structural forms of fluorescein has been proposed in a previous report in relation to the polarity of the solvent and solution pH.45 FITC can be expected to behave in a similar manner because the −NCS group cannot be ionized.46 The proposed structural changes of FITC to different solution environments are shown in Scheme 2.

Scheme 3. Equilibrium Between CTAB As Monomers and CTAB Forming Micelles

CTABmon and CTABmic represent the CTAB molecules in monomer and micelle forms, respectively; n is the total number of surfactant molecules; N is the total number of surfactant molecules used in the formation of micelles; r is the mean aggregation number of micelles; and NA is Avogadro’s number. N/r is the number of micelles formed in the system. K is the micellization equilibrium constant, which is also the ratio of micelle formation constant, kfM to micelle deformation constant, kdM, as shown in eq 1.47

Scheme 2. Different Forms of FITC

K=

kf M kd M

(1)

The cmc may be determined by any changes on the physicochemical properties of the aqueous CTAB solution as a function of the surfactant concentration. As shown in Figure 1, significant changes to the emission characteristics (maximum intensity and wavelength) of FITC are observed at different concentrations of CTAB. To detect precisely the behavioral changes, more than 30 samples were prepared with [CTAB]T ranging from 0 to ≤0.02 M at a fixed concentration of FITC and 1% of ethanol. Figure 2 shows the maximum emission intensity (Imax) as a function of CTAB concentration.

As shown in Scheme 2, the existence of FITC in different forms varies depending on the microenvironment they settle in. The lactonic form dominates in nonpolar solvent, whereas the ionic forms dominate in the polar solvent. The equilibrium between Anion V and Dianion VI is governed by the polarity of the solvent and solution pH. In aqueous solutions at pH ∼6.5, FITC exists predominantly as Dianion VI.46 Throughout the CTAB concentration range used in the current study, the solution pH remained constant at ∼6.2. In addition, the shape of the spectra (Figure 1) also remains the same, suggesting that FITC predominantly exists in Dianion VI form in the absence and presence of CTAB, as indicated in a previous report for a similar probe in aqueous CTAB solutions.45 The intensity maximum, Imax, of the spectrum is directly proportional to the concentration of FITC in the form of Dianion VI. The shift in λmax is related to the more stable binding of FITC in its excited state to the CTAB molecules. A detailed discussion on the second observation (changes to λmax) will be provided later. First, to explore further the suitability of FITC for probing the microstructural properties of CTAB system, the cmc of CTAB was estimated using the changes to Imax values. Surfactant molecules aggregate to form micelles at cmc, as illustrated in Scheme 3. An equilibrium is established between surfactant molecules in bulk solution and those in the micelle.

Figure 2. Fluorescence emission intensities observed at [FITC]T = 1 × 10−5 M and [CTAB]T ranging from 0 to 0.015 M in aqueous solutions containing 1% of ethanol. Inset: Magnification of the plot in the low concentration range of CTAB.

It is clear from the data shown in Figure 2 that two breakpoints occur, termed as BP1 and BP2. The magnification of the trend observed at low concentrations of CTAB is shown as an inset in Figure 2. The Imax decreases with an increase in CTAB concentration until the first BP1, remains almost constant until BP2, increases with a further increase in CTAB concentration from the second breakpoint, BP2, and reaches a plateau at high concentrations. The observed trend is also confirmed at different concentrations of FITC, as shown in Figure 3. The average values of BP1 and BP2 are found to be 0.021 ± 0.006 and 1.35 ± 0.35 mM, respectively. The individual values are also listed in Table 1. Whereas the BP2 occurs at approximately the same CTAB concentration for 0.0005 to 0.002 mM FITC concentrations (significant difference observed at 0.005 mM is due to experimental 15021

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Figure 3. Relative fluorescence intensity data for [FITC]T = 2 × 10−5 M (●), 5 × 10−5 M (▲), and 7 × 10−5 M (■) with [CTAB]T ranging from 0 to ≤0.020 M in aqueous solutions containing 1% of ethanol.

error), the concentration at which the maximum relative intensity is reached varies. The maximum relative intensity is reached at a relatively lower CTAB concentration for the lowest FITC concentration. This is due to the interaction between CTA+ and FITC, which will be discussed later. The use of other fluorescence probes, for example, pyrene, resulted in only one breakpoint at the cmc of CTAB.48 The two breakpoints observed in this study are new and may provide significant information on the CTAB micelle system. CTAB micelle in 100% water has been extensively studied and its cmc has been found to be in the range of 0.96 to 1.1 mM.43,47 This closely agrees with the average BP2 observed in the current study using FITC as the probe, and hence BP2 = cmc of CTAB. A slight difference in the cmc determined using FITC to that reported in the literature may be due to either the presence of 1% ethanol in the solution or an experimental error. Therefore, to support the reliability of this method, we determined the cmc of CTAB by other techniques. In general, to determine the cmc experimentally, a graph of suitable physical property versus surfactant concentration needs to be plotted. The cmc is marked by the abrupt change in the slope. In this study, conductivity and surface tension measurements were carried out. The results are shown in Figure 4a, and b for respective conductivity and surface tension experiments. The conductivity values showed a significant increase with increasing concentrations of CTAB. This increase is caused by an increase in the concentrations of CTA+ and Br− ions in the solution. The conductivity increases until an average value of [CTAB]T = 1.44 mM. Beyond this concentration, the conductivity behaves in a different manner. Whereas the conductivity still increases with an increase in CTAB concentration, the relative increase is lower − the increase in conductivity has a gradient of ∼80 below 1.44 mM and ∼20 above this concentration. The positively charged CTA+ layer of the micelles attracts oppositely charged ions (counterions). The closest counterions attached to this layer, known as the Stern layer, can partially mask the charge by up to 90% of the original

Figure 4. (a) Conductivity data for different [CTAB], [FITC] = (a) 5 × 10−7 M (□), (b) 1 × 10−5 M (○), (c) 2 × 10−5 M (●), (d) 5 × 10−5 M (▲), and (e) 7 × 10−5 M (×) in aqueous system with 1% ethanol. (b) Surface tension data for the same system.

charge. Hence, the ion-conducting ability of the solution as a whole is significantly reduced. With the increasing concentration of CTAB, the equilibrium shifts toward micellized form of CTAB (CTABmic), and at the same time, the CTAB in monomer form (CTABmon) decreases. From this concept, the fractional ionization constant, α, of micelle is calculated by taking the ratio of the gradients observed in conductivity measurements after and before the breakpoint using eq 2. α = m2 /m1

(2)

m2 is the gradient above cmc and m1 is the gradient below cmc. The average value of α is found to be 0.25 ± 0.02, which is in agreement with the literature value within experimental errors.49 This may be rationalized by the fact that the change in the ion conductivity of aqueous ionic CTAB solution below and above cmc is due to the different degree of ionization. When the concentration of CTAB is low, the molecules exist as monomers, which behave as strong electrolytes, whereas at high concentration of CTAB, the micelles are only partially ionized. Therefore, the ion-conducting ability decreases. By comparing the results obtained in the current study to those compiled in the literature43,49−51 on the characteristics of aqueous CTAB micelles, it is confirmed that the presence of FITC has no significant effect on the formation of CTAB micelles. On the basis of the conductivity measurements, the cmc of CTAB is determined to be 1.44 ± 0.2 mM.

Table 1. BP1, BP2, cmc, KM, and α Values Obtained with Different FITC Concentrations in the Studied Sample Condition Using Fluorescence, Conductivity, and Surface Tension Techniques fluorescence measurement [FITC]T (mM)

BP1 (mM)

BP2 (mM)

0.0005 0.01 0.02 0.05 average

0.018 0.019 0.027 0.015 0.021 ± 0.006

1.12 1.00 1.14 2.10 1.35 ± 0.35

conductivity −1

surface tension

KM (M )

cmc (mM)

α

cmc (mM)

± ± ± ± ±

1.15 1.30 1.65 1.65 1.44 ± 0.29

0.23 0.27 0.25 0.24 0.25 ± 0.02

1.00 1.25 1.15 1.30 1.20 ± 0.2

6379 6697 6880 6342 6575

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635 634 460 531 305

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The surface tension measurements were also carried out under exactly the same experimental conditions as those for the conductivity measurements at different concentrations of FITC and CTAB. The results are shown in Figure 4b. From the extrapolated lines of the two very distinguishable slopes, the intersection points were found to be almost of the same values for all different systems, with an average value of 1.2 ± 0.2 mM. CTA+ ions are surface-active due to the presence of a hydrophobic tail; they adsorb at the air−water interface and reduce the surface tension. As the CTAB concentration is increased, more of the CTA+ ions adsorb at the interface, reducing the surface tension further. Once the cmc is reached, CTA+ ions form micelles rather than moving to the air−water interface. Therefore, after the cmc is reached, the interfacial concentration of CTA+ remains constant, leading to approximately constant surface tension values beyond the cmc. The cmc for CTAB from the conductivity and surface tension measurements agrees with the second breakpoint, BP2, measured using the fluorescence technique within experimental errors. The value is also within the range reported in the literature,43,47 as previously mentioned. However, the first breakpoint carries valuable meaning, too, which will be explained later. These experimental data support the fact that FITC can be used as an efficient probe for the determination of cmc of CTAB in aqueous solutions. Effect of FITC Inclusion on the Properties of CTAB Micelle System. Whereas FITC seems to be a suitable probe to determine the cmc of CTAB, one should make sure that the probe is behaving as desired and does not affect the system to be investigated in any way. In particular, the observation of two break points as noted in Figures 2 and 3, prompted us to investigate this system in detail. Therefore, the ionic micelle binding constant of CTAB micelles (KM) in aqueous solutions in the presence of FITC and 1% ethanol was quantified and compared with that of pure CTAB micelles, reported in the literature.43,49−51 It should be noted that the main characteristic of interest is the well-defined, cmc when CTAB monomers start to self-aggregate. As explained in the previous section, the cmc of the micelle system in this study does not deviate significantly from the cmc of CTAB reported in the literature.43,47 KM values at different concentrations of CTAB were calculated using a known equation (eq 3).47,50 Iobs =

I0 W + I0 MKM 1 + KM[Dn]

Figure 5. Fluorescence microscopy images for 0.005 M CTAB solution (left) and 0.015 M CTAB solution (right); [FITC] = 1 × 10−5 M.

microscopy due to the intercalation of FITC molecules with micelles’ Stern layer. A similar experiment was performed at 0.005 M CTAB (image on the left); however, no micelles could be observed, possibly due to the existence of very small spherical micelles. As previously mentioned, fluorescence probes have been used for measuring the cmc of micelles.48 However, because the probes used in these studies were not sensitive enough to the microenvironment changes due to the growing micelles, most of them did not show any changes in the fluorescence characteristics such as the emission intensity.48 With micelle growth processes reported in the literature53 and evidenced from the images shown in Figure 5 (image on the right), we speculate that the micelle structures change from spherical to short rodlike in a specific concentration range of CTAB. In the current study, the growth of micelles (from spherical to short rodlike) is indicated by the increasing FITC emission intensity (Imax) above the cmc (BP2; Figures 2 and 3), which will be discussed later. Such changes to the fluorescence properties of a probe to the microstructural changes in micelles have not been reported in the literature. In addition to the changes in Imax at various CTAB concentrations, it can also be noticed in the fluorescence spectra shown in Figure 1 that there is a slight red shift in the λmax with an increase in CTAB concentration; it reaches a constant value after BP1. For clarification, the maximum emission wavelength is plotted against CTAB concentrations for various FITC concentrations in Figure 6. The shift in λmax is found to be in the range of ∼518−529 nm depending on the CTAB concentration. At CTAB concentrations above the BP2, the λmax remains almost constant. The red shifts observed are somewhat similar to a system involving fluorescein, as reported by Hadjianestis et al.52 and Song et al.46 This shift in λmax might be due to FITC dimer formation or the formation of a new type of micelle due to the interaction between FITC and CTAB micelles or simply due to the electrostatic interaction between FITC and CTAB molecules below the cmc. First, let us consider the possibility of FITC dimer formation as previously detected by the red shift observed.46,52 To investigate further on this issue, we carried out UV−visible absorption measurements in the presence of constant concentration of CTAB and increasing concentration of FITC, and the results are shown in Figure 7a. The emission intensity maximum as a function of FITC is shown in Figure 7b. It can be seen clearly from Figure 7a that the absorption increases linearly with an increase in the FITC concentration. However, the maximum emission intensity does not increase linearly with an increase in FITC concentration, as shown in

(3)

Iobs represents the observed intensity values from fluorescence spectra, I0W and I0M are the fluorescence intensities in the absence and presence of CTAB, respectively, and [Dn] is the concentration of CTAB. The KM values were calculated using a nonlinear least-squares technique47 and listed in Table 1. The values are almost constant and agree with the value reported in the literature.51 The observation of similar KM values in the absence and presence of FITC suggests that the inclusion of FITC does not affect the behavior of the CTAB micelle system. FITC as a Probe to Monitor Microstructural Transitions in CTAB Micelles. The formation and growth of CTAB micelles in aqueous solutions have been extensively studied for decades.52 To provide evidence of the occurrence of structural changes in CTAB micelles, we took the fluorescence images of the solutions below and above the cmc (BP2), and they are shown in Figure 5. In the image on the right, the structures of micelles are visible under fluorescence optical 15023

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Figure 6. Shift in the wavelength maximum (λmax) of fluorescence emission spectrum with (a) [FITC]T = 1 × 10−5, (b) [FITC]T = 2 × 10−5, (c) [FITC]T = 5 × 10−5, and (d) [FITC]T = 7 × 10−5 with [CTAB]T ranging from 0 to ≤0.015 M in aqueous solutions containing 1% ethanol.

Figure 8. UV−visible absorption spectra of FITC with the increasing [FITC]T in 5 mM CTAB aqueous solution containing 1% ethanol.

(i) Broadening of the absorption band (ii) Appearance of a new shoulder band on the red edge (iii) Red shift of the UV−visible absorption spectra However, none of the three characteristics as listed above was observed upon close analysis of the spectra shown in Figure 8. Therefore, it may be concluded that no self-aggregation of FITC occurred in the system. One can also argue that the red shift on the fluorescence emission spectra may be caused by the reabsorption effect of spontaneously emitted photons by optically active ions.59,60 To prove that the observed effects are not due to reasborption effect, similar fluorescence experiments were carried out with FITC concentration limited to 5 × 10−7 M. The results are shown in Figure 9: even with FITC concentration as low as 5 × 10−7 M, a red shift in λmax is observed, eliminating the possibility of reasborption effect. It is possible that the red shift in λmax and the existence of BP1 are caused by the interaction between anionic molecules of FITC and cationic molecules of CTAB as monomers. A similar interaction was reported for a system consisting of CTAB and cyclodextrins, where two cmc’s were observed due to the formation of two types of micelles: pure CTAB micelle and the CTAB−cyclodextrin complex micelle.45 This possibility may fit the current study if the real cmc is reduced to BP1, and BP2 is due to the second type of micelle formed. In a study by Gao,43 the interaction of FITC and CTAB was studied by means of Langmuir adsorption process. The aggregation between CTAB and FITC was thought to be possible when the concentration of CTAB is about the same as its cmc in water either in monomeric or micellized form.46 However, because the BP1 is

Figure 7. (a) Plot showing the UV absorption at band maximum with increasing [FITC]T in the presence of 5 mM CTAB and 1% of ethanol in aqueous solutions. (b) Fluorescence emission intensity maximum under similar experimental conditions as in panel a.

Figure 7b. In fact, the emission intensity showed a maximum at [FITC]T = 0.01 mM and slightly decreased beyond this concentration. This supports the speculation that either there may be increasing interaction between the FITC molecules themselves54−56 or there is a limit in using this probe, which involves the reabsorption effect and self-quenching of the FITC probe molecules.57 Even though the self-aggregation of the probe molecules is a common phenomenon, it may affect the equilibrium between micelle formation (kfM) and micelle deformation processes (kdM). To explore this issue further, we show a closer look at the absorption spectra of the system in Figure 8 for clarification. Figure 8 shows the absorption spectra for selected concentrations of CTAB solutions shown in Figure 7. From the arguments in the literature, in the case where probe interaction (FITC aggregation) occurs, there are a few characteristics that should be visible in the spectra.58 The characteristics are: 15024

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Therefore, the shift in emission wavelength toward a longer wavelength might be due to a stronger binding between FITC in its excited state with the CTAB in micelle form.46 This interaction between FITC excited state and CTAB monomer also leads to a decrease in emission intensity due to the changes in its microenvironment. As previously mentioned, FITC exists in different structural forms depending on the polarity of the microenvironment.37−42 FITC exists mainly as Dianion VI in a polar solvent such as water and as Lactone IV in apolar solvent such as toluene and cyclohexane.45 The structural form of FITC in aqueous solution is illustrated in Scheme 4a with Dianion VI predominating as free ions. With the introduction of cationic CTAB into the system below its cmc, the microenvironment surrounding the dianions is changed, and the dianions are partially stabilized (neutralized) due to the positively charged CTAB monomers. This is illustrated in Scheme 4b. This leads to a decrease in fluorescence intensity and a shift in λmax, which continues up to BP1 and remains constant due to the saturation of the stabilization caused by CTAB monomers. However, after the cmc (BP2), the cationic CTAB molecules start to aggregate together, forming micelles with their polar head/Stern layer on the outer side and hydrophobic tail as the core of the micelle. The anionic nature of FITC and the more polar condition of the Stern layer of the micelle as compared with water causes the concentration of FITC in aqueous phase ([FITC]aq) to decrease due to the shift of the molecule to micelle phase ([FITC]mic). The settling of FITC molecules in the Stern layer of micelle favors the formation of Dianion VI. This is shown by the similar spectral shapes shown in the absence and presence of CTAB (Figure 1). Therefore, the increasing of [FITC] mic leads to an increase in the concentration of Dianion VI in the stern layer. This is illustrated in Scheme 5. As previously mentioned, micelles grow with increasing concentration of the surfactant molecules. The growth also results in the increasing micellized form of FITC ([FITC]mic) in its Dianion VI form. The structural change/ growth results in the increasing of Imax, as observed in this study.

Figure 9. (a) Fluorescence emission data for [FITC]T = 5 × 10−7 M and [CTAB]T ranging from 0 to 0.015 M in aqueous solutions containing 1% ethanol. Inset: Magnification of the plot in the low concentration range of CTAB. (b) Shift in the wavelength maximum (λmax) of fluorescence emission spectrum with increasing [CTAB]T and [FITC]T = 5 × 10−7 M.

10 times lower than the cmc of CTAB in water, it excludes the possibility of BP1 being the critical concentration for FITCCTAB monomers aggregation. In the absence of FITC dimer formation and the occurrence of a second cmc by FITC, it can be suggested that the red shift observed in the fluorescent spectra might be due to an increase in the electrostatic interaction between the anionic FITC and cationic CTAB micelles in its excited state. It is a known phenomenon that the excited state of fluorophore molecules relax to the ground state by fluorescence process.61,62 The stability of the molecule in the excited state may result in a longer lifetime in the excited state, indicated by the λmax. Scheme 4a

a

(a) FITC molecules in Dianion VI form exist as free monomers in polar aqueous system and (b) in the presence of CTAB below its cmc, the micropolarity environment surrounding the FITC is altered due to the presence of CTA+ ions. 15025

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charged micelles at a molecular level. It can also be noted that FITC spectral behavior is very sensitive upon any changes of its microenvironment, even when CTAB concentration is more than 10 times lower than its cmc.

Scheme 5. Illustration Where the FITC Molecules in the Form of Dianion Are Now Intercalated in between Micelle Heads in the Stern Layer of Micelle

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CONCLUSIONS In this Article, we have demonstrated the possibility of using FITC as a fluorescence probe in monitoring micelle aggregation behavior: from a very low concentration of CTAB where they exist as monomers, the micelle formation at cmc, and at high concentrations where the structural growth occurs. In the development of the method as a probe to monitor micelle structural transitions, careful consideration is given to every single detail of the optical properties of FITC. With a change in CTAB concentrations, two breakpoints in Imax and a significant shift in λmax were observed. Three very important issues have been addressed in this study. The first point is the testing of FITC to measure the cmc of CTAB. The second point is the interaction between CTAB and FITC below the cmc. The third and the most important issue is the ability of FITC to probe the micelle growth. It can be concluded from the data shown and discussion provided that the spectral response of FITC toward the increasing concentration CTAB is sensitive enough to probe the formation of CTAB micelles to the growth of spherical micelles and short rodlike micelles. This novel method developed may be useful for studying the structural behaviors of any micelle system in general.

For a clearer understanding, and to summarize the whole concept of relationship between Imax and its microenvironment, the FITC settlement in various CTAB systems is represented in Scheme 6. Scheme 6. Interaction of FITC in Different CTAB Microenvironments

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At low CTAB concentrations, FITC is stabilized in aqueous phase when the CTAB monomers were introduced to the system, resulting in a decrease in Imax up to point BP1 when the stabilization is saturated due to higher concentration of CTAB, [CTAB]mon ≫ [FITC]aq. From the point of micellization of CTAB (BP2; cmc), the [FITC]aq shifts to [FITC]mic, thus increasing the polarity of its microenvironment and increasing Imax. Another interesting trend of Imax upon [CTAB]T as seen in Figure 3 is the decreasing positive slopes with an increase in FITC concentration. A steeper slope observed for the system with the lowest FITC concentration is due to the incorporation of different amounts of FITC in micelle phase at a given CTAB concentration. For the system with a higher concentration of FITC, a relatively lower amount is incorporated in the CTAB due to the shifts in the equilibria, which supports the step-bystep interaction of FITC with CTAB microenvironment, as illustrated by Scheme 6. At higher concentrations of FITC, a higher fraction of FITC will be in its aqueous phase. It has also been previously discussed that [FITC]aq gives a relatively lower emission intensity due to different polar environments in water and micelle phase. Taking these points into account, the equilibrium between [FITC]aq−[CTAB]mon and [FITC]mic− [CTAB]mic shifts to the left, resulting in a slower increase in Imax, as shown in Figure 3. Another comment that can be made is about the changes in the λmax observed in these microenvironments. When present in water (polar environment), the λmax is 518 nm. A red shift is observed when the Dianion VI’s charge is neutralized by CTAB (Scheme 4). When FITC settles in a different kind of polar environment in the stern layer of the micelle, the λmax is different from that observed in pure water. It is clear from the discussion provided that FITC can be used to probe the structural changes in CTAB micelles. The discussion also emphasizes the interaction between the counterion and the

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], masho@unimelb. edu.au. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS N.S.M.Y. and M.N.K. acknowledge the University of Malaya for the award of Bright Sparks-SLAB-SLAI scholarship (N.S.M.Y.) and a grant, UM.C/HIR/MOHE/SC/07. M.A. acknowledges financial support from the Australian Research Council (Discovery Project scheme).

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REFERENCES

(1) Mukerjee, P.; Mysels, K. J. Natl. Stand. Ref. Data Syst. 1971, 230− 233. (2) Chevalier, Y.; Zemb, T. Rep. Prog. Phys. 1990, 53, 279−370. (3) Cordes, E. H.; Gitler, C. Prog. Bioorg. Chem 1973, 2, 1−53. (4) Moulik, S. P. Curr. Sci. 1996, 71, 368−376. (5) Nelson, P. H.; Rutledge, G. C.; Hatton, T. A. J. Phys. Chem. 1997, 107, 10777−10781. (6) Ketner, A. M.; Kumar, R.; Davies, T. S.; Elder, P. W.; Raghavan, S. R. J. Am. Chem. Soc. 2007, 129, 1553−1559. (7) Hassan, P. A.; Raghavan, P. A.; Kaler, E. W. Langmuir 2002, 18, 2543−2548. (8) Ziserman, L.; Abezgauz, L.; Ramon, O.; Raghavan, S. R.; Danino, D. Langmuir 2009, 25, 10483−10489. (9) Davies, T. S.; Ketner, A. M.; Raghavan, S. R. J. Am. Chem. Soc. 2006, 128, 6669−6675. (10) Blanazs, A.; Armes, S. P.; Ryan, A. J. Macromol. Rapid Commun. 2009, 30, 267−277. (11) Yang, J. Curr. Opin. Colloid Interface Sci. 2002, 7, 276−281. (12) Ziserman, L.; Abezgauz, L.; Ramon, O.; Raghavan, S. R.; Danino, D. Langmuir 2009, 25, 10483−10489. 15026

dx.doi.org/10.1021/jp304854h | J. Phys. Chem. C 2012, 116, 15019−15027

The Journal of Physical Chemistry C

Article

(54) Ogawa, M.; Kosaka, N.; Choyke, P. L.; Kobayashi, H. ACS Chem. Biol. 2009, 4, 20892−21088. (55) Johansson, M. K. In Methods in Molecular Biology; Humana Press: Totowa, NJ, 2006; Vol. 335. (56) Johansson, M. K.; Cook, R. M. Chem.Eur. J. 2003, 9, 3466− 3471. (57) Dhami, S.; Mello, A. J. D.; Rumbles, G.; Bishop, S. M.; Philips, D.; Beeby, A. Photochem. Photobiol. 2008, 61, 341−346. (58) Kühn, H.; Petermann, K.; Huber, G. Opt. Lett. 2010, 35, 1524− 1526. (59) Williams, A. T. R.; Winfield, S. A.; Miller, J. N. Analyst 1983, 108, 1067−1071. (60) Dhami, S.; Mello, A. J.; Rumbles, G.; Bishop, S. M.; Phillips, D.; Beeby, A. Photobiology 1995, 61, 341−346. (61) So, P. T. C.; Dong, C. Y. Encyclopedia of Life Sciences;≫ Macmillan Publishers Ltd, Nature Publishing Group, London, 2002. (62) Yardley, J. T.; Specht, L. T. Chem. Phys. Lett. 1975, 37, 543− 546.

(13) Magid, L. J.; Han, Z.; Warr, G. G.; Cassidy, M. A.; Butler, P. D.; Hamilton, W. A. J. Phys. Chem. B 1997, 101, 7919−7927. (14) Aswal, V. K.; Kohlbrecher, J.; Goyal, P. S.; Amenitsh, H.; Bernstorff, S. J. Phys.: Condens. Matter 2006, 18, 11399−11410. (15) Yusof, N. S. M.; Khan, M. N. Langmuir 2010, 26, 10627−10635. (16) Yusof, N. S. M.; Khan, M. N. J. Colloid Interface Sci. 2011, 360, 182−188. (17) Asakawa, T.; Kitano, H.; Ohta, A.; Miyagishi, S. J. Colloid Interface Sci. 2001, 242, 284−287. (18) He, Z. M.; O’Connor, P. J.; Romsted, L. S.; Zanette, D. J. Phys. Chem. 1989, 93, 4219−4226. (19) Zana, R. J. Colloid Interface Sci. 1980, 78, 330−337. (20) Iso, K.; Okada, T. Langmuir 2000, 16, 9199−9204. (21) Aniansson, E. A. G.; Wall, S. N.; Almgren, M.; Hoffmann, H.; Kielmann, I.; Ulbricht, R.; Zana, R.; Lang, J.; Tondre, C. J. Phys. Chem. 1976, 80, 905−922. (22) Candau, S. J.; Hirsch, E.; Zana, R.; Adam, M. J. Colloid Interface Sci. 1988, 122, 430−440. (23) Gelbart, W. M.; Ben-Shaul, A.; Roux, D. Membranes, Microemulsions and Monolayers; Springer: New York, 1994. (24) Israelachvili, J. N. Intermolecular and Surface Forces; Academic Press: London, 1992. (25) Rafati, A. A.; Safatian, F. Phys. Chem. Liq. 2008, 46, 587−598. (26) Afkhami, A.; Khalafi, L. J. Chil. Chem. Soc. 2007, 54, 431−436. (27) Chen, X.-M. Chin. J. Chem. Phys. 2011, 24, 484−488. (28) Rawat, S. S.; Chattopadhyay, A. J. Fluoresc. 1999, 9, 233−244. (29) Ananthapadmanabhan, K. P.; Goddard, E. D.; Turro, N. J.; Kuo, P. L. Langmuir 1985, 1, 352−355. (30) Kida, N. Y.; Nakatsuji, Y.; Akashi, M. Langmuir 2005, 21, 6688− 6695. (31) Smith, L. M.; et al. Methods Enzymol. 1987, 155, 260−301. (32) Szewcyk, B.; Summers, D. F. Anal. Biochem. 1987, 164, 303− 306. (33) Vigers, G. P. A.; et al. J. Cell Biol 1988, 107, 1011−1024. (34) Der-Balian, G. P.; et al. Anal. Biochem. 1988, 173, 59−63. (35) Song, A.; Wu, T.; Chen, S.; Zhang., M.; Shen, T. Dyes Pigm. 1998, 39, 371−382. (36) Song, A.; Wu, T.; Zhang., M.; Shen., T. Chem. Lett. 1998, 321, 907−911. (37) Babcock, D. F.; Kramp, D. C. J. Biol. Chem. 1983, 258, 6380− 6389. (38) Martin, M. M.; Lindqvist, L. Lumin. J. 1975, 10, 381−390. (39) Martin, E.; Pardo, A.; Guijarro, M. S.; Fernandezalonso, J. I. J. Mol. Struct. 1986, 142, 197−200. (40) Zeng, H.-H.; Wang, K.-M.; Yang, X.-H.; Yu, R.-Q. Anal. Chim. Acta 1994, 287, 267−278. (41) Martin, M. M. Chem. Phys. Lett. 1975, 35, 105−111. (42) Martin, M. M. Chem. Phys. Lett. 1976, 43, 332−337. (43) Gao, H.-W.; Zhou, D.-Y. Bull. Korean Chem. Soc. 2002, 23, 29− 33. (44) Ogawa, T.; Aoyagi, S.; Miyasaka, T.; Sakai, K. Sensors 2009, 9, 8271−8277. (45) Zhao, Z.; Shen, T.; Xu, H. Spectrochim. Acta 1989, 45A, 1113− 1116. (46) Song, A.; Zhang, J.; Manhua, Z.; Shen, T.; Tang, J. Colloids. Surf., A 2000, 167, 253−262. (47) Khan, M. N. Micellar Catalysis: Surfactant Science Series; CRC Taylor & Francis: Boca Raton, FL, 2007; Vol. 133. (48) Aguiar, J.; Carpena, P.; Molina-Bolivar, J. A.; Ruiz, C. C. J. Colloid Interface Sci. 2003, 258, 116−122. (49) Valero, M. J. Inclusion Phenom. Macrocyclic Chem. 2002, 42, 121−133. (50) Khan, M. N. J. Phys. Org. Chem. 1996, 9, 295−300. (51) Gao, H.-W.; Zheng, L.-X.; Mei, H.-D. Instrum. Sci. Technol. 2004, 32, 271−280. (52) Hadjianestis, J.; Nikokavouras, J. J. Photochem. Photobiol., A. 1993, 69, 337−343. (53) Berret, J.-F. Mol. Gels 2006, 6, 667−720. 15027

dx.doi.org/10.1021/jp304854h | J. Phys. Chem. C 2012, 116, 15019−15027