Characterization of the Structural Transitions in CTAB Micelles UsingFluorescein IsothiocyanateNor 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 detectthe structural changes that occur in micelle systems. The sensitivity offluorescein isothiocyanate (FITC) has been evaluated for (i) detecting themicellization of cetyltrimethyl ammonium bromide (CTAB) and (ii)probing the concentration dependent aggregation, leading to micro-structural changes that occur within CTAB micelles. The critical micelleconcentration (cmc) of CTAB has been determined to be 1.35 ± 0.35 mMusing the fluorescence spectral characteristics of FITC. Because theexperimental conditions have been altered to optimize FITC probing, thecmc is also validated by surface tension and conductivity measurements. Tomake sure FITC does not affect the properties of micelles, we calculatedthe micelle binding constant, KM, at different concentrations of FITC usinga nonlinear least-squares method. The average KM for [FITC]T ≤ 5 mM isfound to be 6575 ± 233. The optical properties of FITC have also beenfound to be sensitive in response to the changes in the polarity of the microenvironment, caused by the structural changes inCTAB/water system. Two significant observations are noticed from the fluorescence spectra of FITC in CTAB solutions: (i) adecrease 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-dependentmicrostructural changes in CTAB micelles. On the basis of the experimental observations, FITC is found to be a suitablefluorescent probe for monitoring the changes in CTAB micelle structures.

■ INTRODUCTIONMicellization is a reversible self-assembly process of surface-active materials above a well-defined threshold concentration,known as the critical micelle concentration (cmc).1,2 Themorphology of micelles relies heavily on the electrostatic/stericrepulsion between the hydrophilic heads and the attractiveinteractions of the hydrophobic tails. In general, the tail of asurfactant may consist of 8 to 18 carbons.3 The average size ofspherical micelles is ∼50 Å with around 40−100 surfactantmolecules per micelle unit.4,5 However, manipulation of theelectrostatic/steric and the hydrophobic interactions by anymeans may result in the formation of different structures, suchas cylindrical,6,7 wormlike,4,8−10 and vesicles.9,10 Known as the“smart nano-materials” and “living polymers”,11,12 the flexibilityof micelles has attracted people from industries as well as theresearchers to explore the possibilities and advantages theymight 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 electro-chemistry,19 and electrophoresis.20 Micelle structures may besignificantly altered by the slightest changes in experimentalconditions, such as the addition of hydrophobic counterions.The growth of micelles may be induced by a decrease in theelectrostatic repulsion between the micelle heads and an

increase in the hydrophobic interaction between the tails. Suchgrowth may also occur simply due to an increase in theconcentration of the surfactants. Because the changes tomicrostructures during the concentration-dependent growth ofmicelles may be very subtle, the use of a very sensitive probe isrecommended. It is also important to make sure that the probeitself does not significantly affect the process of either micelleaggregation or deformation. This equilibrium has beendescribed in review articles21,22 and textbooks.23,24 Anyinteraction of the probe with surfactant molecules may resultin probe-micelle aggregation, leading to the formation ofdifferent type of “impure” micelles, as reported in a systeminvolving cyclodextrin and CTAB.25−27

Fluorescence probing has been a useful method for thecharacterization of many systems including micelles.28 A searchin the literature results in a variety of fluorophore compoundssensitive in detecting the cmc of surfactants. This is mainlyassociated with the significant changes in the optical propertiesof the fluorophore due to the presence of surface-activemolecules in monomer and micelle forms.29,30 However, afundamental understanding of the suitability of fluorophores for

Received: May 18, 2012Revised: June 19, 2012Published: June 22, 2012

Article

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

monitoring the interaction between micelles as well as thestructural changes that occur above the cmc is still lacking,especially for systems with minimal micelle growth, such asconcentration-dependent microstructural changes.In this study, a novel method has been developed to probe

the microstructural changes that occur in aqueous CTABsolutions using fluorescein isothiocyanate (FITC) as afluorescence probe. FITC is a well-known fluorescent labelfor a variety of systems including proteins, antibodies, andlectins.31−34 It shows sensitive photophysical responses, whichdepend on its microenvironmental properties, such as polar-ity,35,36 solution pH,37−39 and H-bonding.40−42 The variationsin observable responses include the absorption and fluores-cence spectral characteristics and fluorescence quantum yieldand lifetime. The interaction of FITC with CTAB is known tofollow Langmuir monolayer adsorption with 1:3 ratio due tothe negative and positive charges of FITC and CTAB,respectively.43 Furthermore, significant changes in FITCfluorescence spectra were observed with a difference in thepolarity of microenvironment surrounding the probe forproteins and different ionic micelle systems.43,44 This providespromising insight into probing the concentration-dependentstructural changes that occur in CTAB micelles using FITC. Toevaluate the suitability of FITC to determine the micelleproperties of CTAB, we measured the cmc of CTAB andcompared it with that measured by other conventionalmethods. We then used FITC for detecting the changes tothe structural properties of CTAB micelles.

■ MATERIALS AND METHODSMaterials. Cetyltrimethyl ammonium bromide (CTAB)

and fluorescein isothiocyanate isomer I (FITC) werecommercial products of Sigma Aldrich of highest availablepurity and therefore used as received. There are two possibleisomers for FITC: Isomer I with thiocyanate group on the metaposition to the carboxyl group of the benzene ring and IsomerII with thiocyanate group on the para position to the carboxylgroup of the benzene ring, as shown in Scheme 1. Isomer I has

been used in this study because it can be isolated easily in itspure 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 ethanolwas 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 fivedifferent 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 theconcentration range, [CTAB]T = 0 to ≤0.02 M at room

temperature (23.9 ± 1.2 °C). [ ]T is the total concentrations inmicelle and aqueous phases. All samples were sonicated byimmersing them in a sonication bath (45 kHz, ∼300 W, totalvolume capacity 20 L) for 5 min to allow sufficient time forFITC to equilibrate in the desired environment. From theliterature, it is known that the maximum interaction betweenFITC and the surfactants could be reached within 3 min ofstanding time.43 LabCHEM conductivity meter and Analite2141 surface tension meter were used for conductivity andsurface tension experiments, respectively. A negligible change inthe temperature during the course of the experiments did notaffect the conductivity and surface tension values. UV−visibleabsorption spectra of the samples were measured using VarianCary 50 Bio UV−visible spectrophotometer. The fluorescencespectra of the samples were measured using ShimadzuRF5301PC spectrofluorophotometer with the excitation wave-length fixed at 505 nm. Fluorescence spectra were recorded inthe emission range 505−700 nm. Fluorescence images weretaken using Olympus Panasonic IX71 optical/fluorescencemicroscope. All measurements were repeated at least twice.

■ RESULTS AND DISCUSSION

FITC Spectral Response to Changes in CTAB concen-tration (Determination of cmc). Prior to evaluating thesuitability of FITC for monitoring the concentration dependentmicrostructural changes in CTAB micelles, the effect of[CTAB] on the emission properties of FITC was investigated.The fluorescent spectra observed in aqueous solutionscontaining 1 × 10−5 M FITC with varying amounts of CTABare shown in Figure 1. On the basis of the UV−visibleabsorption characteristics, an excitation wavelength of 505 nmwas used to record the fluorescence spectra.The spectral variations observed in Figure 1 demonstrate the

dramatic sensitivity of FITC to the changes in theconcentration of CTAB. A gradual decrease in the maximumintensity is observed for CTAB at low concentrations up to 0.3mM, followed by a significant increase with a further increase in

Scheme 1. Structures of FITC Isomers

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.

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the concentration. This transformation is also accompanied bya 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 solutionenvironments should be considered. Variation in the structuralforms of fluorescein has been proposed in a previous report inrelation to the polarity of the solvent and solution pH.45 FITCcan be expected to behave in a similar manner because the−NCS group cannot be ionized.46 The proposedstructural changes of FITC to different solution environmentsare shown in Scheme 2.

As shown in Scheme 2, the existence of FITC in differentforms varies depending on the microenvironment they settle in.The lactonic form dominates in nonpolar solvent, whereas theionic forms dominate in the polar solvent. The equilibriumbetween Anion V and Dianion VI is governed by the polarity ofthe 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 thecurrent study, the solution pH remained constant at ∼6.2. Inaddition, the shape of the spectra (Figure 1) also remains thesame, suggesting that FITC predominantly exists in Dianion VIform in the absence and presence of CTAB, as indicated in aprevious report for a similar probe in aqueous CTABsolutions.45 The intensity maximum, Imax, of the spectrum isdirectly proportional to the concentration of FITC in the formof Dianion VI. The shift in λmax is related to the more stablebinding of FITC in its excited state to the CTAB molecules. Adetailed discussion on the second observation (changes to λmax)will be provided later. First, to explore further the suitability ofFITC for probing the microstructural properties of CTABsystem, the cmc of CTAB was estimated using the changes toImax values.Surfactant molecules aggregate to form micelles at cmc, as

illustrated in Scheme 3. An equilibrium is established betweensurfactant molecules in bulk solution and those in the micelle.

CTABmon and CTABmic represent the CTAB molecules inmonomer and micelle forms, respectively; n is the total numberof surfactant molecules; N is the total number of surfactantmolecules used in the formation of micelles; r is the meanaggregation number of micelles; and NA is Avogadro’s number.N/r is the number of micelles formed in the system. K is themicellization equilibrium constant, which is also the ratio ofmicelle formation constant, kf

M to micelle deformationconstant, kd

M, as shown in eq 1.47

=Kkk

fM

dM

(1)

The cmc may be determined by any changes on thephysicochemical properties of the aqueous CTAB solution as afunction of the surfactant concentration.As shown in Figure 1, significant changes to the emission

characteristics (maximum intensity and wavelength) of FITCare observed at different concentrations of CTAB. To detectprecisely the behavioral changes, more than 30 samples wereprepared with [CTAB]T ranging from 0 to ≤0.02 M at a fixedconcentration of FITC and 1% of ethanol. Figure 2 shows themaximum emission intensity (Imax) as a function of CTABconcentration.

It is clear from the data shown in Figure 2 that twobreakpoints occur, termed as BP1 and BP2. The magnificationof the trend observed at low concentrations of CTAB is shownas an inset in Figure 2. The Imax decreases with an increase inCTAB concentration until the first BP1, remains almostconstant until BP2, increases with a further increase in CTABconcentration from the second breakpoint, BP2, and reaches aplateau at high concentrations. The observed trend is alsoconfirmed at different concentrations of FITC, as shown inFigure 3. The average values of BP1 and BP2 are found to be0.021 ± 0.006 and 1.35 ± 0.35 mM, respectively. Theindividual values are also listed in Table 1. Whereas the BP2occurs at approximately the same CTAB concentration for0.0005 to 0.002 mM FITC concentrations (significantdifference observed at 0.005 mM is due to experimental

Scheme 2. Different Forms of FITC

Scheme 3. Equilibrium Between CTAB As Monomers andCTAB Forming Micelles

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

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error), the concentration at which the maximum relativeintensity is reached varies. The maximum relative intensity isreached at a relatively lower CTAB concentration for the lowestFITC concentration. This is due to the interaction betweenCTA+ 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 twobreakpoints observed in this study are new and may providesignificant information on the CTAB micelle system. CTABmicelle in 100% water has been extensively studied and its cmchas been found to be in the range of 0.96 to 1.1 mM.43,47 Thisclosely agrees with the average BP2 observed in the currentstudy using FITC as the probe, and hence BP2 = cmc of CTAB.A slight difference in the cmc determined using FITC to thatreported in the literature may be due to either the presence of1% ethanol in the solution or an experimental error. Therefore,to support the reliability of this method, we determined thecmc of CTAB by other techniques. In general, to determine thecmc experimentally, a graph of suitable physical property versussurfactant concentration needs to be plotted. The cmc ismarked by the abrupt change in the slope. In this study,conductivity and surface tension measurements were carriedout. The results are shown in Figure 4a, and b for respectiveconductivity and surface tension experiments.The conductivity values showed a significant increase with

increasing concentrations of CTAB. This increase is caused byan increase in the concentrations of CTA+ and Br− ions in thesolution. The conductivity increases until an average value of[CTAB]T = 1.44 mM. Beyond this concentration, theconductivity behaves in a different manner. Whereas theconductivity still increases with an increase in CTABconcentration, the relative increase is lower − the increase inconductivity has a gradient of ∼80 below 1.44 mM and ∼20above this concentration. The positively charged CTA+ layer ofthe micelles attracts oppositely charged ions (counterions). Theclosest counterions attached to this layer, known as the Sternlayer, can partially mask the charge by up to 90% of the original

charge. Hence, the ion-conducting ability of the solution as awhole is significantly reduced. With the increasing concen-tration of CTAB, the equilibrium shifts toward micellized formof CTAB (CTABmic), and at the same time, the CTAB inmonomer form (CTABmon) decreases. From this concept, thefractional ionization constant, α, of micelle is calculated bytaking the ratio of the gradients observed in conductivitymeasurements after and before the breakpoint using eq 2.

α = m /m2 1 (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 inagreement with the literature value within experimentalerrors.49 This may be rationalized by the fact that the changein the ion conductivity of aqueous ionic CTAB solution belowand above cmc is due to the different degree of ionization.When the concentration of CTAB is low, the molecules exist asmonomers, which behave as strong electrolytes, whereas at highconcentration of CTAB, the micelles are only partially ionized.Therefore, the ion-conducting ability decreases. By comparingthe results obtained in the current study to those compiled inthe literature43,49−51 on the characteristics of aqueous CTABmicelles, it is confirmed that the presence of FITC has nosignificant effect on the formation of CTAB micelles. On thebasis of the conductivity measurements, the cmc of CTAB isdetermined to be 1.44 ± 0.2 mM.

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 rangingfrom 0 to ≤0.020 M in aqueous solutions containing 1% of ethanol.

Table 1. BP1, BP2, cmc, KM, and α Values Obtained with Different FITC Concentrations in the Studied Sample Condition UsingFluorescence, Conductivity, and Surface Tension Techniques

fluorescence measurement conductivity surface tension

[FITC]T (mM) BP1 (mM) BP2 (mM) KM (M−1) cmc (mM) α cmc (mM)

0.0005 0.018 1.12 6379 ± 635 1.15 0.23 1.000.01 0.019 1.00 6697 ± 634 1.30 0.27 1.250.02 0.027 1.14 6880 ± 460 1.65 0.25 1.150.05 0.015 2.10 6342 ± 531 1.65 0.24 1.30average 0.021 ± 0.006 1.35 ± 0.35 6575 ± 305 1.44 ± 0.29 0.25 ± 0.02 1.20 ± 0.2

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.

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The surface tension measurements were also carried outunder exactly the same experimental conditions as those for theconductivity measurements at different concentrations of FITCand CTAB. The results are shown in Figure 4b. From theextrapolated lines of the two very distinguishable slopes, theintersection points were found to be almost of the same valuesfor all different systems, with an average value of 1.2 ± 0.2 mM.CTA+ ions are surface-active due to the presence of ahydrophobic tail; they adsorb at the air−water interface andreduce the surface tension. As the CTAB concentration isincreased, 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−waterinterface. Therefore, after the cmc is reached, the interfacialconcentration of CTA+ remains constant, leading to approx-imately 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 experimentalerrors. The value is also within the range reported in theliterature,43,47 as previously mentioned. However, the firstbreakpoint carries valuable meaning, too, which will beexplained later. These experimental data support the fact thatFITC can be used as an efficient probe for the determination ofcmc of CTAB in aqueous solutions.Effect of FITC Inclusion on the Properties of CTAB

Micelle System. Whereas FITC seems to be a suitable probeto determine the cmc of CTAB, one should make sure that theprobe is behaving as desired and does not affect the system tobe investigated in any way. In particular, the observation of twobreak points as noted in Figures 2 and 3, prompted us toinvestigate this system in detail. Therefore, the ionic micellebinding constant of CTAB micelles (KM) in aqueous solutionsin the presence of FITC and 1% ethanol was quantified andcompared with that of pure CTAB micelles, reported in theliterature.43,49−51 It should be noted that the main characteristicof interest is the well-defined, cmc when CTAB monomers startto self-aggregate. As explained in the previous section, the cmcof the micelle system in this study does not deviate significantlyfrom the cmc of CTAB reported in the literature.43,47

KM values at different concentrations of CTAB werecalculated using a known equation (eq 3).47,50

=+

+I

I I KK D1 [ ]obs

0W

0M

M

M n (3)

Iobs represents the observed intensity values from fluorescencespectra, I0

W and I0M are the fluorescence intensities in the

absence and presence of CTAB, respectively, and [Dn] is theconcentration of CTAB. The KM values were calculated using anonlinear least-squares technique47 and listed in Table 1. Thevalues are almost constant and agree with the value reported inthe literature.51 The observation of similar KM values in theabsence and presence of FITC suggests that the inclusion ofFITC does not affect the behavior of the CTAB micelle system.FITC as a Probe to Monitor Microstructural Tran-

sitions in CTAB Micelles. The formation and growth ofCTAB micelles in aqueous solutions have been extensivelystudied for decades.52 To provide evidence of the occurrence ofstructural changes in CTAB micelles, we took the fluorescenceimages of the solutions below and above the cmc (BP2), andthey are shown in Figure 5. In the image on the right, thestructures of micelles are visible under fluorescence optical

microscopy due to the intercalation of FITC molecules withmicelles’ Stern layer. A similar experiment was performed at0.005 M CTAB (image on the left); however, no micelles couldbe observed, possibly due to the existence of very smallspherical micelles.As previously mentioned, fluorescence probes have been

used for measuring the cmc of micelles.48 However, because theprobes used in these studies were not sensitive enough to themicroenvironment changes due to the growing micelles, mostof them did not show any changes in the fluorescencecharacteristics such as the emission intensity.48 With micellegrowth processes reported in the literature53 and evidencedfrom the images shown in Figure 5 (image on the right), wespeculate that the micelle structures change from spherical toshort rodlike in a specific concentration range of CTAB. In thecurrent study, the growth of micelles (from spherical to shortrodlike) is indicated by the increasing FITC emission intensity(Imax) above the cmc (BP2; Figures 2 and 3), which will bediscussed later. Such changes to the fluorescence properties of aprobe to the microstructural changes in micelles have not beenreported in the literature.In addition to the changes in Imax at various CTAB

concentrations, it can also be noticed in the fluorescencespectra shown in Figure 1 that there is a slight red shift in theλmax with an increase in CTAB concentration; it reaches aconstant value after BP1. For clarification, the maximumemission wavelength is plotted against CTAB concentrationsfor 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 concen-trations above the BP2, the λmax remains almost constant. Thered shifts observed are somewhat similar to a system involvingfluorescein, as reported by Hadjianestis et al.52 and Song et al.46

This shift in λmax might be due to FITC dimer formation or theformation of a new type of micelle due to the interactionbetween FITC and CTAB micelles or simply due to theelectrostatic interaction between FITC and CTAB moleculesbelow the cmc.First, let us consider the possibility of FITC dimer formation

as previously detected by the red shift observed.46,52 Toinvestigate further on this issue, we carried out UV−visibleabsorption measurements in the presence of constantconcentration of CTAB and increasing concentration ofFITC, and the results are shown in Figure 7a. The emissionintensity maximum as a function of FITC is shown in Figure7b.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 increaselinearly with an increase in FITC concentration, as shown in

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

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Figure 7b. In fact, the emission intensity showed a maximum at[FITC]T = 0.01 mM and slightly decreased beyond thisconcentration. This supports the speculation that either theremay be increasing interaction between the FITC moleculesthemselves54−56 or there is a limit in using this probe, whichinvolves the reabsorption effect and self-quenching of the FITCprobe molecules.57 Even though the self-aggregation of theprobe molecules is a common phenomenon, it may affect theequilibrium between micelle formation (kf

M) and micelledeformation processes (kd

M). To explore this issue further,we show a closer look at the absorption spectra of the system inFigure 8 for clarification.Figure 8 shows the absorption spectra for selected

concentrations of CTAB solutions shown in Figure 7. Fromthe arguments in the literature, in the case where probeinteraction (FITC aggregation) occurs, there are a fewcharacteristics that should be visible in the spectra.58 Thecharacteristics are:

(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 abovewas observed upon close analysis of the spectra shown in Figure8. Therefore, it may be concluded that no self-aggregation ofFITC occurred in the system. One can also argue that the redshift on the fluorescence emission spectra may be caused by thereabsorption effect of spontaneously emitted photons byoptically active ions.59,60 To prove that the observed effectsare not due to reasborption effect, similar fluorescenceexperiments were carried out with FITC concentration limitedto 5 × 10−7 M. The results are shown in Figure 9: even withFITC concentration as low as 5 × 10−7 M, a red shift in λmax isobserved, 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 ofFITC and cationic molecules of CTAB as monomers. A similarinteraction was reported for a system consisting of CTAB andcyclodextrins, where two cmc’s were observed due to theformation of two types of micelles: pure CTAB micelle and theCTAB−cyclodextrin complex micelle.45 This possibility may fitthe current study if the real cmc is reduced to BP1, and BP2 isdue to the second type of micelle formed. In a study by Gao,43

the interaction of FITC and CTAB was studied by means ofLangmuir adsorption process. The aggregation between CTABand FITC was thought to be possible when the concentrationof CTAB is about the same as its cmc in water either inmonomeric or micellized form.46 However, because the BP1 is

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

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

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10 times lower than the cmc of CTAB in water, it excludes thepossibility of BP1 being the critical concentration for FITC-CTAB 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 shiftobserved in the fluorescent spectra might be due to an increasein the electrostatic interaction between the anionic FITC andcationic CTAB micelles in its excited state. It is a knownphenomenon that the excited state of fluorophore moleculesrelax to the ground state by fluorescence process.61,62 Thestability of the molecule in the excited state may result in alonger lifetime in the excited state, indicated by the λmax.

Therefore, the shift in emission wavelength toward a longerwavelength might be due to a stronger binding between FITCin its excited state with the CTAB in micelle form.46 Thisinteraction between FITC excited state and CTAB monomeralso leads to a decrease in emission intensity due to the changesin 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 aswater and as Lactone IV in apolar solvent such as toluene andcyclohexane.45 The structural form of FITC in aqueoussolution is illustrated in Scheme 4a with Dianion VIpredominating as free ions. With the introduction of cationicCTAB into the system below its cmc, the microenvironmentsurrounding the dianions is changed, and the dianions arepartially stabilized (neutralized) due to the positively chargedCTAB monomers. This is illustrated in Scheme 4b. This leadsto a decrease in fluorescence intensity and a shift in λmax, whichcontinues up to BP1 and remains constant due to the saturationof the stabilization caused by CTAB monomers.However, after the cmc (BP2), the cationic CTAB molecules

start to aggregate together, forming micelles with their polarhead/Stern layer on the outer side and hydrophobic tail as thecore of the micelle. The anionic nature of FITC and the morepolar condition of the Stern layer of the micelle as comparedwith water causes the concentration of FITC in aqueous phase([FITC]aq) to decrease due to the shift of the molecule tomicelle phase ([FITC]mic). The settling of FITC molecules inthe Stern layer of micelle favors the formation of Dianion VI.This is shown by the similar spectral shapes shown in theabsence and presence of CTAB (Figure 1). Therefore, theincreasing of [FITC]mic leads to an increase in theconcentration of Dianion VI in the stern layer. This isillustrated in Scheme 5. As previously mentioned, micelles growwith increasing concentration of the surfactant molecules. Thegrowth 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 thisstudy.

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

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, themicropolarity environment surrounding the FITC is altered due to the presence of CTA+ ions.

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For a clearer understanding, and to summarize the wholeconcept of relationship between Imax and its microenvironment,the FITC settlement in various CTAB systems is represented inScheme 6.

At low CTAB concentrations, FITC is stabilized in aqueousphase when the CTAB monomers were introduced to thesystem, resulting in a decrease in Imax up to point BP1 when thestabilization is saturated due to higher concentration of CTAB,[CTAB]mon ≫ [FITC]aq. From the point of micellization ofCTAB (BP2; cmc), the [FITC]aq shifts to [FITC]mic, thusincreasing the polarity of its microenvironment and increasingImax. Another interesting trend of Imax upon [CTAB]T as seen inFigure 3 is the decreasing positive slopes with an increase inFITC concentration. A steeper slope observed for the systemwith the lowest FITC concentration is due to the incorporationof different amounts of FITC in micelle phase at a given CTABconcentration. For the system with a higher concentration ofFITC, a relatively lower amount is incorporated in the CTABdue to the shifts in the equilibria, which supports the step-by-step interaction of FITC with CTAB microenvironment, asillustrated by Scheme 6. At higher concentrations of FITC, ahigher fraction of FITC will be in its aqueous phase. It has alsobeen previously discussed that [FITC]aq gives a relatively loweremission intensity due to different polar environments in waterand micelle phase. Taking these points into account, theequilibrium between [FITC]aq−[CTAB]mon and [FITC]mic−[CTAB]mic shifts to the left, resulting in a slower increase inImax, as shown in Figure 3.Another comment that can be made is about the changes in

the λmax observed in these microenvironments. When present inwater (polar environment), the λmax is 518 nm. A red shift isobserved when the Dianion VI’s charge is neutralized by CTAB(Scheme 4). When FITC settles in a different kind of polarenvironment in the stern layer of the micelle, the λmax isdifferent from that observed in pure water. It is clear from thediscussion provided that FITC can be used to probe thestructural changes in CTAB micelles. The discussion alsoemphasizes the interaction between the counterion and the

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

■ CONCLUSIONSIn this Article, we have demonstrated the possibility of usingFITC as a fluorescence probe in monitoring micelle aggregationbehavior: from a very low concentration of CTAB where theyexist as monomers, the micelle formation at cmc, and at highconcentrations where the structural growth occurs. In thedevelopment of the method as a probe to monitor micellestructural transitions, careful consideration is given to everysingle detail of the optical properties of FITC. With a change inCTAB concentrations, two breakpoints in Imax and a significantshift in λmax were observed. Three very important issues havebeen addressed in this study. The first point is the testing ofFITC to measure the cmc of CTAB. The second point is theinteraction between CTAB and FITC below the cmc. The thirdand the most important issue is the ability of FITC to probe themicelle growth. It can be concluded from the data shown anddiscussion provided that the spectral response of FITC towardthe increasing concentration CTAB is sensitive enough toprobe the formation of CTAB micelles to the growth ofspherical micelles and short rodlike micelles. This novelmethod developed may be useful for studying the structuralbehaviors of any micelle system in general.

■ AUTHOR INFORMATION

Corresponding Author*E-mail: nsyusof@student.unimelb.edu.au, masho@unimelb.edu.au.

NotesThe authors declare no competing financial interest.

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

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

Scheme 6. Interaction of FITC in Different CTABMicroenvironments

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