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Effect of counterion on the structural switchover and binding of piroxicam with sodium dodecyl sulfate (SDS) micelles
Journal of Colloid and Interface Science 292 (2005) 265–270 www.elsevier.com/locate/jcis
Effect of counterion on the structural switchover and binding of piroxicam with sodium dodecyl sulfate (SDS) micelles Hirak Chakraborty, Munna Sarkar ∗ Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Calcutta 700064, India Received 28 March 2005; accepted 19 May 2005 Available online 3 August 2005
Abstract Addition of an electrolyte such as NaCl to ionic micelles such as sodium dodecyl sulfate (SDS) alters the ionic atmosphere of the bulk solvent, thereby changing both the micellar properties and the interaction pattern of micelles with a molecule in the solvent. In this study, we show how added NaCl in the presence of SDS micelles modulates the surface charge of the micelles, which in turn fine-tunes the switchover equilibrium between anionic and global neutral forms of piroxicam. The presence of salt alters the CMC and aggregation number of SDS micelles. The binding of the global neutral form of piroxicam with SDS is found to be strongly modulated by the presence of counterion in the bulk solvent. 2005 Elsevier Inc. All rights reserved. Keywords: Surfactants; Salt effect; Switchover; Apparent pKa ; Change in free energy (G)
1. Introduction Surface-active agents (surfactants) are classified by the nature of their hydrophilic head groups and hydrophobic tails. The nature of both the hydrophobic tails and hydrophilic head groups controls the physical properties of the micelles, viz., the critical micellar concentration (CMC) and aggregation number (n) [1]. Since the head group is the link between the micelle and the bulk solvent, it plays the principal role in guiding the behavior of the micelles with the bulk solvent as well as with any molecule present in the solvent. Addition of a neutral electrolyte such as NaCl, KCl, or NaBr to the ionic micelles (SDS, CTAB, etc.) results in a decrease in the CMC value and an increase in the aggregation number [2]. The decrease in the CMC value is mainly due to the decrease in the thickness of the ionic atmosphere surrounding the ionic head groups in the presence of the additional electrolyte and the consequent decreased electrical repulsion between them in the micelles [1]. The extent of this effect * Corresponding author. Fax: +91 33 23374637.
E-mail address: [email protected] (M. Sarkar). 0021-9797/$ – see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2005.05.056
is ion-specific; the smaller the radius of the hydrated ion, the greater is the effect. Effectiveness in decreasing CMC + + + follows the order NH+ 4 > K > Na > Li . Increase in aggregation number is a consequence of a salting-out phenomenon in which the added salt ties up water molecules, resulting in an increase in the effective concentration of surfactant, thereby increasing the aggregation number [3]. The presence of electrolyte not only changes the physical properties of the micelles but also changes the interaction pattern of micelles with a molecule in the bulk solvent. The counterion also enters the interfacial region and modifies it. Piroxicam [4-hydroxy-2-methyl-N -(pyridin-2-yl)–2H1,2-benzothiazine-3-carboxamide 1,1-dioxide] is a very interesting molecule to both chemists and biochemists. For biochemists the molecule is interesting because of its multifunctional activity, including chemoprevention [4], chemosuppression [5], and UV sensitization [6]. For the chemist, the molecule is interesting for its structural sensitivity to the environment, which is a unique feature of this drug [7]. The molecule exists in different structural forms such as anionic, zwitterionic, and neutral (Fig. 1), depending on the nature of its immediate environment [8]. Despite the fact
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2. Materials and methods
Fig. 1. Structure of different prototropic forms of piroxicam.
that piroxicam has two dissociable sites, spectroscopically the zwitterionic and neutral forms are indistinguishable. This limits the description of the dissociation constant in terms of microscopic species. The zwitterionic and neutral forms are collectively called the “global neutral” form; they exist in equilibrium with the anionic form in solution [9]. In the presence of micelles, piroxicam can switch over or convert from the global neutral form to the anionic form and vice versa. This switchover is modulated and fine-tuned by the surface charge of the micelles [9]. The sensitivity of the switchover has been correlated with the change in apparent pKa values with varying micellar parameters [9]. Addition of counterions in a micellar medium will affect not only micellar parameters but also the ionic environment near the surface of the micelles. In order to understand the effect of the counterion on the equilibrium between the global neutral and the anionic forms of piroxicam in the presence of SDS micelles, we have studied the interaction of piroxicam with SDS in the absence and in the presence of various concentrations of NaCl. In addition, we have studied the effect of counterions on the binding of the global neutral form of piroxicam with SDS micelles. To do so, we carried out our experiments at pH 1.8, where only the global neutral form will exist in solution. We have first characterized the micellar medium at pH 1.8 and in presence of different concentrations of NaCl by optical spectroscopic techniques. The CMC values were measured using the intrinsic fluorescence of piroxicam. The aggregation number of SDS micelles at pH 1.8 in the absence and in the presence of different NaCl concentrations was measured by the method of quenching of pyrene fluorescence by cetylpyridinium chloride (CPC) [10]. Our present study clearly demonstrates how the presence of counterions affects the structural switchover of piroxicam and the binding of the global neutral form of piroxicam with SDS micelles.
Piroxicam was purchased from Sigma Chemicals (USA) and was used without further purification. Sodium dodecyl sulfate (SDS) and sodium chloride (NaCl) were purchased from Merck, Germany and SRL, India, respectively. Water was glass-distilled three times before use. Stock solutions of piroxicam of concentration 0.5 mM were prepared in ethanol (Merck, Germany) and the exact concentration was adjusted by triple distilled water. Each aliquot contains a maximum of 6% (v/v) of ethanol. We have changed the pH of the working solutions by adding dilute HCl to them. The volume of acid (HCl) added to the working solutions is exactly equal to the volume of acid that is needed to acidify a volume of water equal to the working solution to attain that particular pH. Samples were checked for photochemical changes during spectral scan time and no change was found. We kept the surfactant concentration well above the CMC value during the determination of pKa to ensure the presence of an adequate number of micelles in the solution, to keep the micellar effect uniform at every pH. The concentration of piroxicam was kept constant at 30 µM for all samples. Fluorescence lifetimes were determined from total emission intensity decay measurements, using a time-resolved fluorimeter assembled in our laboratory with components from Edinburgh Analytical Instruments (EIA, UK) and EG& G ORTEC (USA) and operated in the time-correlated-singlephoton-counting mode. A pulsed high-pressure (1.5-atm) N2 lamp operating at repetition rate 25 kHz was used as a source. The pulse profile had a full width at half maximum (FWHM) of 1.2 ns. Pyrene was excited by the 337 nm N2 line and its emission was monitored at 393 nm. Slits with bandpass 16 nm were used in both excitation and emission channels. Intensity decay curves could be fitted to a single exponential series, I (t) = A0 + A1 exp(−t/τ1 ), where A1 is the pre-exponential factor representing the contribution of the time-resolved decay of the component with a lifetime τ1 . A0 is a constant. The decay parameters were obtained using a software package supplied by EIA, which uses a Marquardt iterative nonlinear least squares fitting procedure. Statistically the goodness of the fit was evaluated by the reduced χ 2 value. Absorption spectra were recorded with a Themo Spectronic spectrophotometer, Model UNICAM UV500. Baseline correction was done with water before each set of data were recorded. Fluorescence measurements were performed using a Hitachi spectrofluorimeter, Model 4010. All emission spectra were corrected for instrument response at each wavelength. A 2 mm × 10 mm path length quartz cell was used for all fluorescence measurements to avoid any blue edge distortion of the spectrum due to inner filter effect [11]. All measurements were done with freshly prepared samples and at a constant temperature of 298 K.
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3. Results and discussion 3.1. Effect of NaCl on the N ↔ A switchover equilibrium In our earlier studies we have shown that the surface charge of the micelles modulates the micelle-induced switchover between the global neutral (N) and anionic (A) forms of piroxicam [8]. Doping the uniformly charged micelles with even a small amount of oppositely charged head group was capable of fine-tuning this N and A equilibrium [12]. The underlying cause behind the switchover between two prototropic forms of piroxicam was found to be the change in the apparent pKa value in the presence of different micelles [8]. In this work we have determined the pKa values in the presence of a constant concentration of SDS micelles with different NaCl concentrations. This work was done by measuring the ratio of the concentration of the anion to the neutral forms at different pH from the absorbance values at 363 and 335 nm, respectively (Fig. 2a).
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The extinction coefficients used for the anion and the global neutral forms are 2.43 × 104 and 3.52 × 104 M−1 cm−1 , respectively [13]. The point where [A]/[N] = 1 is considered to be the apparent pKa of piroxicam in the absence and in the presence of various concentrations of NaCl. It should be noted that we have kept the micellar concentration at 12 mM, which is well above the CMC values in the entire range of pH variation studies. Hence the shift in pKa value reflects the effect of NaCl on the dissociation of piroxicam in the presence of fully formed micelles. The pKa value in the absence of NaCl is 3.29 and in the presence of 0.05, 0.10, 0.15, and 0.20 mol L−1 of NaCl the pKa values are 2.91, 2.81, 2.71, and 2.65, respectively. Decrease in pKa with increased concentration of NaCl indicates that SDS micelles do not favor the global neutral form of piroxicam in the presence of an elevated concentration of the counterion. With the addition of NaCl, more and more Na+ counterions assemble near the negatively charged surface of the micelles and occupy the interface, thereby screening the negative charge. As the micellar surface is screened by the presence of the Na+ counterions, the negatively charged SDS micelles loses its preference for the global neutral form of piroxicam with increasing NaCl concentration. Hence the N ↔ A equilibrium shifts to the right as compared to that in the presence of SDS micelles with no added NaCl. In order to see how addition of NaCl would affect the N ↔ A equilibrium that is induced by micelle formation, we have calculated the change in free energy using the standard equation G = −RT ln K.
Fig. 2. (a) Trace of [A]/[N] of piroxicam in SDS micellar medium in the presence of various concentrations of NaCl (0.0 [2], 0.05 [1], 0.10 [a], 0.15 [P], and 0.20 mol L−1 ["]) with pH; (b) variation of G (J/mol) with concentration of SDS at different concentrations of NaCl (0.0 [2], 0.05 [1], 0.10 [a], 0.15 [P], and 0.20 mol L−1 ["]) at pH 2.64.
Here K represents the equilibrium constant of N ↔ A conversion that is induced by micelle formation at a constant pH. In this case we monitor the change in free energy at a constant pH with increasing surfactant concentration. The same experiment was repeated for various concentrations of NaCl to determine how NaCl modulates micelle-induced N ↔ A equilibrium. Fig. 2b shows the plot of G vs concentration of SDS in the absence and in the presence of various concentrations of NaCl. The change in G means the difference between G values at the highest concentration of the surfactant and in the absence of surfactant. For this study pH was kept constant at 2.64 to keep the initial conditions similar for all sets of experiments. The changes in G values are −2.53, −2.29, −2.12, −2.02, and −1.99 kJ mol−1 in the absence and in the presence of 0.05, 0.1, 0.15, and 0.2 mol L−1 NaCl, respectively. The value decreases with increased NaCl concentration, indicating that the formation of the global neutral form is less favored. Our results clearly show that any modulation of the surface charge of micelles is capable of fine-tuning the switchover equilibrium between N and A. In this case, the modulation of the surface charge of SDS micelles is achieved by the charge-screening effect of Na+ counterions.
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Fig. 3. (a) Plot of relative fluorescence intensity of the global neutral form of piroxicam monitored at 475 nm by exciting at 335 nm with the concentration of SDS at pH 1.8 in the presence of different concentration of NaCl (0.0 [2], 0.05 [1], 0.10 [a], 0.15 [P], and 0.20 mol L−1 ["]); (b) plot of log CCMC of SDS at different concentrations of NaCl vs log CNaCl at pH 1.8; (c) plot of aggregation number (!) and excited state lifetime of pyrene (") in SDS micelles with increasing concentration of NaCl.
3.2. Binding of neutral form of piroxicam with SDS micelles at various NaCl concentrations In SDS micelles, it is the global neutral form of piroxicam that gets incorporated [8]. In order to see how the binding of global neutral form of piroxicam is affected by counterions, experiments were carried out at pH 1.8 when only the global neutral form is present. It is important to see how NaCl would affect micellar parameters at this pH (1.8) before binding of global neutral form of piroxicam is considered. 3.3. Determination of CMC and aggregation number Critical micellar concentrations of SDS micelles at pH 1.8 in the absence and in the presence of various concentration of NaCl were determined by using the intrinsic fluorescence properties of piroxicam. The relative fluorescence intensity of the global neutral form of piroxicam, monitored at 475 nm by exciting at 335 nm, increases with increasing concentration of SDS at pH 1.8 as shown in Fig. 3a. The inflection
point of the plot of relative fluorescence intensity vs concentration of SDS gives the value of the CMC. The CMC values of SDS at pH 1.8 as determined from Fig. 3a are 3.71 × 10−3 , 2.01 × 10−3 , 1.63 × 10−3 , 1.48 × 10−3 , and 1.33 × 10−3 mol L−1 in the absence and in the presence of 0.05, 0.1, 0.15, and 0.2 mol L−1 NaCl, respectively. The decrease in CMC value in the presence of NaCl, as expected, follows the linear equation [1] log CCMC = −a log Ci + b, where Ci is the concentration of the total monovalent counterion and a and b are constants having the values 0.292 and −3.076, respectively (Fig. 3b). The aggregation number of the SDS micelles at pH 1.8 in the absence and in the presence of NaCl was determined by exploiting the fluorescence quenching of pyrene by cetylpyridinium chloride (CPC) [10]. The measured ratio of fluorescence intensity of pyrene monitored at 393 nm in the presence of CPC to that in the absence of CPC (F /F0 ) is related to the micellar concentration ([M]) by the expres-
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Table 1 Aggregation number of SDS micelles at pH 1.8 in the absence and in the presence of various concentrations of NaCl and the corresponding excited-state lifetimes of pyrene Concentration of NaCl (mol L−1 )
Aggregation number at 65 × 10−3 mol L−1 SDS
Aggregation number at 75 × 10−3 mol L−1 SDS
Average aggregation number (n)
Excited-state lifetime of pyrene (ns)
0 0.05 0.1 0.15 0.2
95 98 105 109 110
101 102 107 111 122
98 100 106 110 116
152.3 159.5 162.1 167.5 173.6
sion [13] F /F0 = e−([CPC]/[M]) . On the basis of the pseudophase model and provided that [S] CMC, where [S] is the stoichiometry concentration of the surfactant, we get ln(F0 /F ) = n[CPC]/[S] by neglecting higher order terms [14]. n is the aggregation number. We have kept the surfactant concentration fixed at 75 × 10−3 mol L−1 and the concentration of CPC was varied. Aggregation number (n) was obtained from the slope of the linear curve when ln(F0 /F ) was plotted against the concentration of CPC. The same experiments have also been performed with 65×10−3 mol L−1 SDS and the average n value (see Table 1) is used for data analysis. Fig. 3b shows that with increased NaCl concentration, the aggregation number increases (Fig. 3c) and the critical micellar concentration decreases. This is explained by the general rule in aqueous solution: the greater the dissimilarity between the solvent and surfactant, the greater is the aggregation number and the lower is the CMC. Addition of NaCl results in the water molecules being tied up by the salt. This increases the effective concentration of the surfactant, thereby promoting aggregation, which leads to an increase in aggregation number with a subsequent decrease in CMC values. The excited state lifetime of pyrene also increases with increased NaCl concentration (Table 1) and parallels the increase in aggregation number (Fig. 3c). This shows that there is an increase in the compactness of the micellar structure with increased aggregation number. The binding constant of the global neutral form of piroxicam with SDS micelles in the absence and in the presence of NaCl at pH 1.8 was measured by following the method described by Almgren et al. [15], 1 F∞ − F0 =1+ , Fc − F0 Kb [M] where F0 , Fc , and F∞ are the fluorescence intensity of the neutral form of piroxicam considered in absence of surfactant, at an intermediate concentration, and in a condition of complete micellization, respectively, Kb being the binding constant and [M] the micellar concentration. The values of
Fig. 4. Plot of (F∞ − F0 )/(Fc − F0 ) vs [M]−1 in the presence of various concentrations of NaCl (0.0 [2], 0.05 [1], 0.10 [a], 0.15 [P], and 0.20 mol L−1 ["]).
n in the determination of micellar concentration have been taken from our pyrene fluorescence quenching data as shown in Table 1. The binding constant (Kb ) values have been determined from the slope of the linear plot of (F∞ − F0 )/(Fc − F0 ) against 1/[M] (Fig. 4). Linear regression analyses were done and the correlation coefficients were found to be greater than or equal to 0.96 for all cases. The calculated Kb values are 1.89 × 105 , 1.67 × 105 , 1.63 × 105 , 1.30 × 105 , and 1.0 × 105 M−1 in the absence and in the presence of 0.05, 0.1, 0.15, and 0.2 mol L−1 NaCl, respectively (intercept is greater than 0.94 for all concentrations of NaCl, which is very close to unity). The difference in the binding constant values may be attributed to the amount of counterion present in the hydration layer of the micelles. As the concentration of Na+ increases in the vicinity of the micellar surface, the negative charge of the micellar head group is screened. This results in the binding constant of the global neutral form decreasing with increased Na+ concentration.
4. Conclusion Addition of counterions such as Na+ to negatively charged micelles such as SDS results in surface charge screening. This not only modulates micellar parameters such
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as CMC and aggregation number but also affects the micelleinduced switchover equilibrium between the global neutral and anionic forms of piroxicam in the presence of SDS micelles. The binding of the global neutral form of piroxicam to the micelles is strongly affected by the presence of NaCl in the bulk aqueous phase.
[2] [3] [4] [5] [6] [7] [8]
Acknowledgments
[9]
The authors are extremely thankful to Professor Subhash Chandra Bhattacharya of Jadavpur University, Kolkata, for his kind gift of CPC. We also thank Rona Banerjee and Sujata Roy of our laboratory for their kind cooperation.
[10]
References
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[1] M.J. Rosen, Surfactants and Interfacial Phenomena, Wiley–Interscience, New York, 1978.
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N.J. Turro, P.L. Kuo, J. Phys. Chem. 90 (1986) 837. N. Erdinc, S. Gokturk, M. Tuncay, J. Pharm. Sci. 93 (2004) 1566. S.R. Ritland, S.J. Gendler, Carcinogenesis 20 (1999) 51. A.P. Goldman, W.E. Longo, N. Panesar, J.E. Mazuski, D.L. Kaminski, Carcinogenesis 19 (1998) 2195. M. Gebhardt, U. Wollina, Z. Rheumatol. 54 (1995) 405. J. Bordner, P.D. Hammen, E.B. Whipple, J. Am. Chem. Soc. 111 (1989) 6572. J.M. Geckle, D.M. Rescek, E.B. Whipple, Magn. Reson. Chem. 27 (1989) 150. H. Chakraborty, R. Banerjee, M. Sarkar, Biophys. Chem. 104 (2003) 315. S. Miyagishi, W. Akasohu, T. Hashimoto, T. Asakawa, J. Colloid Interface Sci. 184 (1996) 527. J.R. Lakowicz, Principles of Fluorescence Spectroscopy, second ed., Kluwer Academic/Plenum, New York, 1999. H. Chakraborty, M. Sarkar, Langmuir 20 (2004) 3551. R. Banerjee, H. Chakraborty, M. Sarkar, Spectrochim. Acta A Mol. Biomol. Spectrosc. 59 (2003) 1213. S. Bhattacharya, H.T. Das, S.P. Moulik, J. Photochem. Photobiol. A Chem. 71 (1993) 257. M. Almgren, F. Grieser, J.K. Thomas, J. Am. Chem. Soc. 101 (1979) 279.
Effect of counterion on the structural switchover and binding of piroxicam with sodium dodecyl sulfate (SDS) micelles Hirak Chakraborty, Munna Sarkar ∗ Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Calcutta 700064, India Received 28 March 2005; accepted 19 May 2005 Available online 3 August 2005
Abstract Addition of an electrolyte such as NaCl to ionic micelles such as sodium dodecyl sulfate (SDS) alters the ionic atmosphere of the bulk solvent, thereby changing both the micellar properties and the interaction pattern of micelles with a molecule in the solvent. In this study, we show how added NaCl in the presence of SDS micelles modulates the surface charge of the micelles, which in turn fine-tunes the switchover equilibrium between anionic and global neutral forms of piroxicam. The presence of salt alters the CMC and aggregation number of SDS micelles. The binding of the global neutral form of piroxicam with SDS is found to be strongly modulated by the presence of counterion in the bulk solvent. 2005 Elsevier Inc. All rights reserved. Keywords: Surfactants; Salt effect; Switchover; Apparent pKa ; Change in free energy (G)
1. Introduction Surface-active agents (surfactants) are classified by the nature of their hydrophilic head groups and hydrophobic tails. The nature of both the hydrophobic tails and hydrophilic head groups controls the physical properties of the micelles, viz., the critical micellar concentration (CMC) and aggregation number (n) [1]. Since the head group is the link between the micelle and the bulk solvent, it plays the principal role in guiding the behavior of the micelles with the bulk solvent as well as with any molecule present in the solvent. Addition of a neutral electrolyte such as NaCl, KCl, or NaBr to the ionic micelles (SDS, CTAB, etc.) results in a decrease in the CMC value and an increase in the aggregation number [2]. The decrease in the CMC value is mainly due to the decrease in the thickness of the ionic atmosphere surrounding the ionic head groups in the presence of the additional electrolyte and the consequent decreased electrical repulsion between them in the micelles [1]. The extent of this effect * Corresponding author. Fax: +91 33 23374637.
E-mail address: [email protected] (M. Sarkar). 0021-9797/$ – see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2005.05.056
is ion-specific; the smaller the radius of the hydrated ion, the greater is the effect. Effectiveness in decreasing CMC + + + follows the order NH+ 4 > K > Na > Li . Increase in aggregation number is a consequence of a salting-out phenomenon in which the added salt ties up water molecules, resulting in an increase in the effective concentration of surfactant, thereby increasing the aggregation number [3]. The presence of electrolyte not only changes the physical properties of the micelles but also changes the interaction pattern of micelles with a molecule in the bulk solvent. The counterion also enters the interfacial region and modifies it. Piroxicam [4-hydroxy-2-methyl-N -(pyridin-2-yl)–2H1,2-benzothiazine-3-carboxamide 1,1-dioxide] is a very interesting molecule to both chemists and biochemists. For biochemists the molecule is interesting because of its multifunctional activity, including chemoprevention [4], chemosuppression [5], and UV sensitization [6]. For the chemist, the molecule is interesting for its structural sensitivity to the environment, which is a unique feature of this drug [7]. The molecule exists in different structural forms such as anionic, zwitterionic, and neutral (Fig. 1), depending on the nature of its immediate environment [8]. Despite the fact
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2. Materials and methods
Fig. 1. Structure of different prototropic forms of piroxicam.
that piroxicam has two dissociable sites, spectroscopically the zwitterionic and neutral forms are indistinguishable. This limits the description of the dissociation constant in terms of microscopic species. The zwitterionic and neutral forms are collectively called the “global neutral” form; they exist in equilibrium with the anionic form in solution [9]. In the presence of micelles, piroxicam can switch over or convert from the global neutral form to the anionic form and vice versa. This switchover is modulated and fine-tuned by the surface charge of the micelles [9]. The sensitivity of the switchover has been correlated with the change in apparent pKa values with varying micellar parameters [9]. Addition of counterions in a micellar medium will affect not only micellar parameters but also the ionic environment near the surface of the micelles. In order to understand the effect of the counterion on the equilibrium between the global neutral and the anionic forms of piroxicam in the presence of SDS micelles, we have studied the interaction of piroxicam with SDS in the absence and in the presence of various concentrations of NaCl. In addition, we have studied the effect of counterions on the binding of the global neutral form of piroxicam with SDS micelles. To do so, we carried out our experiments at pH 1.8, where only the global neutral form will exist in solution. We have first characterized the micellar medium at pH 1.8 and in presence of different concentrations of NaCl by optical spectroscopic techniques. The CMC values were measured using the intrinsic fluorescence of piroxicam. The aggregation number of SDS micelles at pH 1.8 in the absence and in the presence of different NaCl concentrations was measured by the method of quenching of pyrene fluorescence by cetylpyridinium chloride (CPC) [10]. Our present study clearly demonstrates how the presence of counterions affects the structural switchover of piroxicam and the binding of the global neutral form of piroxicam with SDS micelles.
Piroxicam was purchased from Sigma Chemicals (USA) and was used without further purification. Sodium dodecyl sulfate (SDS) and sodium chloride (NaCl) were purchased from Merck, Germany and SRL, India, respectively. Water was glass-distilled three times before use. Stock solutions of piroxicam of concentration 0.5 mM were prepared in ethanol (Merck, Germany) and the exact concentration was adjusted by triple distilled water. Each aliquot contains a maximum of 6% (v/v) of ethanol. We have changed the pH of the working solutions by adding dilute HCl to them. The volume of acid (HCl) added to the working solutions is exactly equal to the volume of acid that is needed to acidify a volume of water equal to the working solution to attain that particular pH. Samples were checked for photochemical changes during spectral scan time and no change was found. We kept the surfactant concentration well above the CMC value during the determination of pKa to ensure the presence of an adequate number of micelles in the solution, to keep the micellar effect uniform at every pH. The concentration of piroxicam was kept constant at 30 µM for all samples. Fluorescence lifetimes were determined from total emission intensity decay measurements, using a time-resolved fluorimeter assembled in our laboratory with components from Edinburgh Analytical Instruments (EIA, UK) and EG& G ORTEC (USA) and operated in the time-correlated-singlephoton-counting mode. A pulsed high-pressure (1.5-atm) N2 lamp operating at repetition rate 25 kHz was used as a source. The pulse profile had a full width at half maximum (FWHM) of 1.2 ns. Pyrene was excited by the 337 nm N2 line and its emission was monitored at 393 nm. Slits with bandpass 16 nm were used in both excitation and emission channels. Intensity decay curves could be fitted to a single exponential series, I (t) = A0 + A1 exp(−t/τ1 ), where A1 is the pre-exponential factor representing the contribution of the time-resolved decay of the component with a lifetime τ1 . A0 is a constant. The decay parameters were obtained using a software package supplied by EIA, which uses a Marquardt iterative nonlinear least squares fitting procedure. Statistically the goodness of the fit was evaluated by the reduced χ 2 value. Absorption spectra were recorded with a Themo Spectronic spectrophotometer, Model UNICAM UV500. Baseline correction was done with water before each set of data were recorded. Fluorescence measurements were performed using a Hitachi spectrofluorimeter, Model 4010. All emission spectra were corrected for instrument response at each wavelength. A 2 mm × 10 mm path length quartz cell was used for all fluorescence measurements to avoid any blue edge distortion of the spectrum due to inner filter effect [11]. All measurements were done with freshly prepared samples and at a constant temperature of 298 K.
H. Chakraborty, M. Sarkar / Journal of Colloid and Interface Science 292 (2005) 265–270
3. Results and discussion 3.1. Effect of NaCl on the N ↔ A switchover equilibrium In our earlier studies we have shown that the surface charge of the micelles modulates the micelle-induced switchover between the global neutral (N) and anionic (A) forms of piroxicam [8]. Doping the uniformly charged micelles with even a small amount of oppositely charged head group was capable of fine-tuning this N and A equilibrium [12]. The underlying cause behind the switchover between two prototropic forms of piroxicam was found to be the change in the apparent pKa value in the presence of different micelles [8]. In this work we have determined the pKa values in the presence of a constant concentration of SDS micelles with different NaCl concentrations. This work was done by measuring the ratio of the concentration of the anion to the neutral forms at different pH from the absorbance values at 363 and 335 nm, respectively (Fig. 2a).
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The extinction coefficients used for the anion and the global neutral forms are 2.43 × 104 and 3.52 × 104 M−1 cm−1 , respectively [13]. The point where [A]/[N] = 1 is considered to be the apparent pKa of piroxicam in the absence and in the presence of various concentrations of NaCl. It should be noted that we have kept the micellar concentration at 12 mM, which is well above the CMC values in the entire range of pH variation studies. Hence the shift in pKa value reflects the effect of NaCl on the dissociation of piroxicam in the presence of fully formed micelles. The pKa value in the absence of NaCl is 3.29 and in the presence of 0.05, 0.10, 0.15, and 0.20 mol L−1 of NaCl the pKa values are 2.91, 2.81, 2.71, and 2.65, respectively. Decrease in pKa with increased concentration of NaCl indicates that SDS micelles do not favor the global neutral form of piroxicam in the presence of an elevated concentration of the counterion. With the addition of NaCl, more and more Na+ counterions assemble near the negatively charged surface of the micelles and occupy the interface, thereby screening the negative charge. As the micellar surface is screened by the presence of the Na+ counterions, the negatively charged SDS micelles loses its preference for the global neutral form of piroxicam with increasing NaCl concentration. Hence the N ↔ A equilibrium shifts to the right as compared to that in the presence of SDS micelles with no added NaCl. In order to see how addition of NaCl would affect the N ↔ A equilibrium that is induced by micelle formation, we have calculated the change in free energy using the standard equation G = −RT ln K.
Fig. 2. (a) Trace of [A]/[N] of piroxicam in SDS micellar medium in the presence of various concentrations of NaCl (0.0 [2], 0.05 [1], 0.10 [a], 0.15 [P], and 0.20 mol L−1 ["]) with pH; (b) variation of G (J/mol) with concentration of SDS at different concentrations of NaCl (0.0 [2], 0.05 [1], 0.10 [a], 0.15 [P], and 0.20 mol L−1 ["]) at pH 2.64.
Here K represents the equilibrium constant of N ↔ A conversion that is induced by micelle formation at a constant pH. In this case we monitor the change in free energy at a constant pH with increasing surfactant concentration. The same experiment was repeated for various concentrations of NaCl to determine how NaCl modulates micelle-induced N ↔ A equilibrium. Fig. 2b shows the plot of G vs concentration of SDS in the absence and in the presence of various concentrations of NaCl. The change in G means the difference between G values at the highest concentration of the surfactant and in the absence of surfactant. For this study pH was kept constant at 2.64 to keep the initial conditions similar for all sets of experiments. The changes in G values are −2.53, −2.29, −2.12, −2.02, and −1.99 kJ mol−1 in the absence and in the presence of 0.05, 0.1, 0.15, and 0.2 mol L−1 NaCl, respectively. The value decreases with increased NaCl concentration, indicating that the formation of the global neutral form is less favored. Our results clearly show that any modulation of the surface charge of micelles is capable of fine-tuning the switchover equilibrium between N and A. In this case, the modulation of the surface charge of SDS micelles is achieved by the charge-screening effect of Na+ counterions.
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Fig. 3. (a) Plot of relative fluorescence intensity of the global neutral form of piroxicam monitored at 475 nm by exciting at 335 nm with the concentration of SDS at pH 1.8 in the presence of different concentration of NaCl (0.0 [2], 0.05 [1], 0.10 [a], 0.15 [P], and 0.20 mol L−1 ["]); (b) plot of log CCMC of SDS at different concentrations of NaCl vs log CNaCl at pH 1.8; (c) plot of aggregation number (!) and excited state lifetime of pyrene (") in SDS micelles with increasing concentration of NaCl.
3.2. Binding of neutral form of piroxicam with SDS micelles at various NaCl concentrations In SDS micelles, it is the global neutral form of piroxicam that gets incorporated [8]. In order to see how the binding of global neutral form of piroxicam is affected by counterions, experiments were carried out at pH 1.8 when only the global neutral form is present. It is important to see how NaCl would affect micellar parameters at this pH (1.8) before binding of global neutral form of piroxicam is considered. 3.3. Determination of CMC and aggregation number Critical micellar concentrations of SDS micelles at pH 1.8 in the absence and in the presence of various concentration of NaCl were determined by using the intrinsic fluorescence properties of piroxicam. The relative fluorescence intensity of the global neutral form of piroxicam, monitored at 475 nm by exciting at 335 nm, increases with increasing concentration of SDS at pH 1.8 as shown in Fig. 3a. The inflection
point of the plot of relative fluorescence intensity vs concentration of SDS gives the value of the CMC. The CMC values of SDS at pH 1.8 as determined from Fig. 3a are 3.71 × 10−3 , 2.01 × 10−3 , 1.63 × 10−3 , 1.48 × 10−3 , and 1.33 × 10−3 mol L−1 in the absence and in the presence of 0.05, 0.1, 0.15, and 0.2 mol L−1 NaCl, respectively. The decrease in CMC value in the presence of NaCl, as expected, follows the linear equation [1] log CCMC = −a log Ci + b, where Ci is the concentration of the total monovalent counterion and a and b are constants having the values 0.292 and −3.076, respectively (Fig. 3b). The aggregation number of the SDS micelles at pH 1.8 in the absence and in the presence of NaCl was determined by exploiting the fluorescence quenching of pyrene by cetylpyridinium chloride (CPC) [10]. The measured ratio of fluorescence intensity of pyrene monitored at 393 nm in the presence of CPC to that in the absence of CPC (F /F0 ) is related to the micellar concentration ([M]) by the expres-
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Table 1 Aggregation number of SDS micelles at pH 1.8 in the absence and in the presence of various concentrations of NaCl and the corresponding excited-state lifetimes of pyrene Concentration of NaCl (mol L−1 )
Aggregation number at 65 × 10−3 mol L−1 SDS
Aggregation number at 75 × 10−3 mol L−1 SDS
Average aggregation number (n)
Excited-state lifetime of pyrene (ns)
0 0.05 0.1 0.15 0.2
95 98 105 109 110
101 102 107 111 122
98 100 106 110 116
152.3 159.5 162.1 167.5 173.6
sion [13] F /F0 = e−([CPC]/[M]) . On the basis of the pseudophase model and provided that [S] CMC, where [S] is the stoichiometry concentration of the surfactant, we get ln(F0 /F ) = n[CPC]/[S] by neglecting higher order terms [14]. n is the aggregation number. We have kept the surfactant concentration fixed at 75 × 10−3 mol L−1 and the concentration of CPC was varied. Aggregation number (n) was obtained from the slope of the linear curve when ln(F0 /F ) was plotted against the concentration of CPC. The same experiments have also been performed with 65×10−3 mol L−1 SDS and the average n value (see Table 1) is used for data analysis. Fig. 3b shows that with increased NaCl concentration, the aggregation number increases (Fig. 3c) and the critical micellar concentration decreases. This is explained by the general rule in aqueous solution: the greater the dissimilarity between the solvent and surfactant, the greater is the aggregation number and the lower is the CMC. Addition of NaCl results in the water molecules being tied up by the salt. This increases the effective concentration of the surfactant, thereby promoting aggregation, which leads to an increase in aggregation number with a subsequent decrease in CMC values. The excited state lifetime of pyrene also increases with increased NaCl concentration (Table 1) and parallels the increase in aggregation number (Fig. 3c). This shows that there is an increase in the compactness of the micellar structure with increased aggregation number. The binding constant of the global neutral form of piroxicam with SDS micelles in the absence and in the presence of NaCl at pH 1.8 was measured by following the method described by Almgren et al. [15], 1 F∞ − F0 =1+ , Fc − F0 Kb [M] where F0 , Fc , and F∞ are the fluorescence intensity of the neutral form of piroxicam considered in absence of surfactant, at an intermediate concentration, and in a condition of complete micellization, respectively, Kb being the binding constant and [M] the micellar concentration. The values of
Fig. 4. Plot of (F∞ − F0 )/(Fc − F0 ) vs [M]−1 in the presence of various concentrations of NaCl (0.0 [2], 0.05 [1], 0.10 [a], 0.15 [P], and 0.20 mol L−1 ["]).
n in the determination of micellar concentration have been taken from our pyrene fluorescence quenching data as shown in Table 1. The binding constant (Kb ) values have been determined from the slope of the linear plot of (F∞ − F0 )/(Fc − F0 ) against 1/[M] (Fig. 4). Linear regression analyses were done and the correlation coefficients were found to be greater than or equal to 0.96 for all cases. The calculated Kb values are 1.89 × 105 , 1.67 × 105 , 1.63 × 105 , 1.30 × 105 , and 1.0 × 105 M−1 in the absence and in the presence of 0.05, 0.1, 0.15, and 0.2 mol L−1 NaCl, respectively (intercept is greater than 0.94 for all concentrations of NaCl, which is very close to unity). The difference in the binding constant values may be attributed to the amount of counterion present in the hydration layer of the micelles. As the concentration of Na+ increases in the vicinity of the micellar surface, the negative charge of the micellar head group is screened. This results in the binding constant of the global neutral form decreasing with increased Na+ concentration.
4. Conclusion Addition of counterions such as Na+ to negatively charged micelles such as SDS results in surface charge screening. This not only modulates micellar parameters such
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as CMC and aggregation number but also affects the micelleinduced switchover equilibrium between the global neutral and anionic forms of piroxicam in the presence of SDS micelles. The binding of the global neutral form of piroxicam to the micelles is strongly affected by the presence of NaCl in the bulk aqueous phase.
[2] [3] [4] [5] [6] [7] [8]
Acknowledgments
[9]
The authors are extremely thankful to Professor Subhash Chandra Bhattacharya of Jadavpur University, Kolkata, for his kind gift of CPC. We also thank Rona Banerjee and Sujata Roy of our laboratory for their kind cooperation.
[10]
References
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