Determination of the Critical Micelle Concentration of Triton X100 Using the Compound 3-( benzoxazol-2-yl )-7-( N , N-diethylamino ) chromen-2one as Fluorescent Probe

In the present study we estimated the Critical Micelle Concentration (CMC) of Triton X-100 in an aqueous buffered medium (PBS buffer, pH 7.4), using 3-(benzoxazol-2-yl)-7-(N,N-diethylamino)chromen-2one (BDC), a push-pull compound, as extrinsic fluorescence probe. The CMC value found, 0.33 mmol L, shows good agreement with data from literature obtained using the fluorescence probe technique. Additionally, the polarity, in terms of the ET(30) scale, and the viscosity of the micelle microenvironment in which the probe is preferentially allocated, was estimated as being 46.9 kcal mol and 70.3 cP, respectively, confirming that this microdomain is polar and highly viscous, with characteristics of polyethylene glycol groups consisting in the palisade layer at the micelle-water interface.


INTRODUCTION
Surfactants are hierarchical materials that have gained increasing interest because of their applications, for example as templates to host and guide the growth of aligned and ordered materials with adjustable pore size, high surface areas, large pore volumes, alternative pore shapes, and controllable framework compositions [1][2][3][4][5][6][7][8][9].Triton X-100 alone or combined with another drive agent has been extensively used as templates in the synthesis of mesoporous materials with catalytic activity, aiming different applications [9][10].
The Critical Micelle Concentration (CMC) is probably the simplest way of describing the colloid and surface behavior of a surfactant solution, being an important parameter for the description of this class of materials.When the concentration of surfactant exceeds the CMC, a micellar system is formed [9,11], which generally has several applications [1,2,9].
The CMC can be determined by measuring changes in physical properties [12][13][14][15].A simple and efficient approach for CMC determination consists in the use of an appropriate spectroscopic probe or a noninvasive spectroscopic methodology, using the intrinsic fluorescence naturally present within the micellar systems, to study the changes in the electronic absorption or emission energies and/or intensities [14][15][16][17].
In the present study, the compound 3-(benzoxazol-2-yl)-7-(N,N-diethylamino)chromen-2one (BDC), Figure 1, was used as extrinsic fluorescence probe to determine, by steady-state fluorescence, the Critical Micelle Concentration of Triton X-100 (TX-100) in phosphate-buffered saline (PBS buffer, pH 7.4) aqueous medium.Additionally, the polarity, expressed in terms of the empirical solvatochromic scale ET(30) [22] and the viscosity of the microenvironment in which the probe is preferentially allocated, was also estimated.

MATERIAL AND METHODS
Triton X-100 (polyethylene glycol tertoctylphenyl ether; n = 9-10, MW ≈ 625.0 g mol -1 , Union Carbide), a nonionic surfactant, Figure 2, was used without any treatment.Similarly, a stock solution of Triton X-100 (36 mmol dm -3 ) was prepared using buffered deionized water (phosphate buffer, PBS, pH 7.4, with a NaCl content of 0.9% (w/w)) [24][25][26].Aliquots of this solution were used to prepare solutions containing 5.0 x 10 -6 mol dm -3 of BDC and different concentrations of Triton X-100, ranging between 0 and 3.6 mmol L -1 .The solutions were prepared in such a way to keep the absorbance of the probe below 0.100 at the excitation wavelength of the probe (442 nm) in order to avoid light reabsorption effects in the fluorescence measurements.
The UV-Vis absorption and emission spectra were recorded using, respectively, a Shimadzu-2501 PC spectrophotometer and a Hitachi F-4500 spectrofluorimeter.
For the fluorescence measurements the slits (excitation/emission) were both fixed in 2.5 nm and the photomultiplier (PMT) voltage set at 700 V.All fluorescence measurements were done at 293 K using a 10 mm square cuvette in a right-angle configuration.
The fluorescence quantum yields of BDC in different media were estimated from the corrected fluorescence spectra, using methodology proposed by Eaton [27].The compound 9,10-diphenylanthracene in cyclohexane (F = 0.90 at 293 K) was employed as fluorescence standard.The profiles of the fluorescence spectra in DMF and in TX-100 micelles, and their respective quantum yields, are very similar, Table 1.This suggests that BDC maintains intense polar interactions in the microdomains in which is preferentially allocated in the TX-100 micelles.This is corroborated by the ET (30) [22] of these microdomains, estimated as being 46.89 kcal mol -1 .It should be emphasized that this value was estimated from the F  estimated for BDC allocated in TX100 micelles, using the linear correlation between F  and ET (30) proposed in [20].This result shows a good agreement with the reported in a previous study involving this same probe in two other surfactants (SDS and CTAB), where it was concluded that BDC tends to be allocated in a viscous, polar and hydrophilic region [20].Despite the presence of water in this microdomain, the high viscosity of the medium should compensate at least partially the suppression of fluorescence caused by this solvent.Similar to the observed in CTAB and SDS, the microenvironment in which BDC is allocated in TX-100 possesses a high viscosity, estimated as being 70.26  0.02 cP, a value little smaller than the viscosity of PEG 400 [28].This value was estimated from a plot of the dependence of ΦF with the viscosity of glycerol/water mixtures, proposed in [20].Thus, this environment is sufficiently viscous to minimize the mobility of BDC, making difficult the decay of this probe in the excited state by other routes than the fluorescence.According to Kumbhakar et al., one of the reasons for the high viscosity in this microenvironment, also known as palisade layer, is the hydration of ions, especially cations, since water molecules in this region tends to undergo a kind of clustering [29].

RESULTS AND DISCUSSION
It is noteworthy that the ET(30) of different glycols decreases as size of the chain increases [22].Based on this information, the ET(30) of the palisade layer of TX-100 should be typical of oligomers based on glycol.In other words, and considering the shape proposed for TX-100 micelles [12], BDC tends to occupy polar microdomains consisting of polyethylene glycol groups (n = 9-10), near the micelle-water interface.Calculated using the absorption and emission maxima, expressed in wavenumber; b Ref. [19]; c Ref. [20]; d Considering the linear correlation between F and ET(30), as shown in Ref. [20].
Figure 4 presents the dependence between the fluorescence spectra of BDC and the concentration of TX-100.
As shown in Figure 4a, the increasing presence of TX-100 aggregates tends to magnify the BDC fluorescence, when compared to what occurs in water, Table 1.As the concentration of TX-100 increases, the rise in the population of pre-aggregates, aggregates and micelles favors the incorporation of BDC in the palisade layers, intensifying the fluorescence.Figure 4b evidences this: the rise of the ratio I/I0 as the concentration of TX-100 increases, with a change in the rate when the concentration of micelles predominates, followed by the stabilization of this ratio.Above the CMC, where BDC is predominantly associated to the micelles, the fluorescence is relatively intense.The very small blue shift of the emission maxima (about 113 cm -1 ) that occurs as BDC fluorescence intensity increases from its solvation only in PBS buffer until TX-100 micelles, reinforces the proposition that the probe tends to migrate to the palisade layers of TX-100, as previously discussed.
The CMC of TX-100 was estimated from the analysis of the graph of the second derivative of the ratio between fluorescence intensity and concentration of the probe,  2 (I/I0)/C 2 , versus concentration of TX-100, Figure 5.The value found (0.33 mmol L -1 ) shows a very good agreement with the reported in the literature, especially in determinations using the fluorescence probe technique.For example, Ruiz and Aguiar also found 0.33 mmol dm -3 , using this technique [12].Karimi et al., demonstrating the potential application of surface plasmon resonance, estimated a CMC of 0.27 mmol L -1 for TX-100 in aqueous medium, using silver nanoparticles as probe [14].Jaiswal et al., exploring the intrinsic fluorescence of TX-100 in water, found a CMC of 0.29 mmol L -1 , while using two different extrinsic fluorescent probes (C-153, or 8-Trifluoromethyl-2,3,5,6-4H-1,H-11-oxa-3a-azabenzo[de]anthracen-10-one, and ANS, or 8anilinonaphthalene-1-sulfonic acid), found respectively, 0.24 and 0.27 mmol L -1 [15].The same authors, using Isothermal Titration Calorimetry, estimated a CMC of 0.21 mmol L -1 [15].

CONCLUSION
In the present study the Critical Micelle Concentration (CMC) of the surfactant Triton X-100 was estimated using the extrinsic fluorescence probe 3-(benzoxazol-2-yl)-7-(N,N-diethylamino) chromen-2-one (BDC), in aqueous and buffered (PBS buffer, pH 7.4) medium.The value found, 0.33 mmol L -1 agrees well with the reported in the literature, principally in determinations using the fluorescence probe technique.The observed behavior by BDC confirms the usefulness of this compound as fluorescent probe.The steady-state fluorescence measurements confirms that the microdomain in which BDC is preferentially allocated in Triton X-100 micelles (the palisade layer) is considerably polar (46.9 kcal mol -1 ) and highly viscous (70.3 cP).This high viscosity is probably a consequence of the hydration of the ions from the buffered medium in the palisade layer, since water molecules in this microdomain tend to be as clusters.

Figure 3
Figure 3 presents the fluorescence spectra of BDC in TX-100 micelles, N,N-Dimethylformamide (DMF) and chloroform.

Table 1 .
Spectroscopic data for BDC in micelles and some different solvents: absorption and emission wavelengths, Stokes' shifts, fluorescence quantum yields, and the empirical solvatochromic parameter ET(30) estimated for the different media.