Physicochemical Studies on the Interaction of Gelatin with Cationic

May 7, 2008 - B formation, coacervation (association of GSm. B) led to add turbidity. With increasing addition of CTAB, the coacervates became disinte...
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J. Phys. Chem. B 2008, 112, 6609–6619

6609

Physicochemical Studies on the Interaction of Gelatin with Cationic Surfactants Alkyltrimethylammonium Bromides (ATABs) with Special Focus on the Behavior of the Hexadecyl Homologue Debolina Mitra, Subhas C. Bhattacharya, and Satya P. Moulik* Centre for Surface Science, Department of Chemistry, JadaVpur UniVersity, Kolkata-700 032, India ReceiVed: October 12, 2007; ReVised Manuscript ReceiVed: February 25, 2008

The interaction of a denatured interfacially active protein, gelatin (G) (at pH 9, above its isoelectric pH 4.84, and ionic strength µ ) 0.005), with a cationic amphiphile, hexadecyl (or cetyl) trimethylammonium bromide, CTAB, has been elaborately studied using a variety of techniques. Two types of protein-surfactant complexes at a concentration below the normal critical micellar concentration (cmc) were formed in solution. The first, G-CTAB (monomer) combined complex (GSIn) adsorbed at the air/solution interface, followed by its gradual B transformation to the poor interfacially active second G-CTAB (aggregate) complex (GSm ) at a critical aggregation concentration (cac) of the interacting oppositely charged surfactant. In the higher concentration B B range, upon completion of GSm formation, coacervation (association of GSm ) led to add turbidity. With increasing addition of CTAB, the coacervates became disintegrated and ultimately remained dissolved in the free micellar solution of CTAB. The above features were studied using the techniques of tensiometry, conductometry, turbidimetry, fluorimetry, and microcalorimetry. The interaction features were prominent at [G] g 0.05 g %, and several of these were either marginal or absent at [G] < 0.05 g %. The denatured protein was found to form viscous as well as gel-forming consistencies at higher [G] and at lower temperature. A temperature variation study on the interaction of G with CTAB has revealed that enhanced interaction takes place at higher temperature. The effect of [G] on its interaction with cationic surfactants of varying chain length in the alkyltrimethylammonium bromide (ATAB) series has been also studied; a similar interactional profile as that of CTAB has been exhibited by octadecyltrimethylammonium bromide; however, the lower homologues (dodecyl- and tetradecyl-) of ATAB have offered different profiles. It has been found that the ATABs with higher alkyl chain lengths were more interactive with negatively charged G than their lower homologues. Quantification of the results in terms of different transition points, counterion binding of the protein-bound surfactant aggregates and free micelles, the enthalpy of binding interactions and energetics of ATAB micellization, and so forth have been studied. The results have been rationalized in terms of an interaction model. Introduction Bio- and synthetic polymer-surfactant (lipid) interactions have relevance in cosmetics and body care product formulations in pharmaceuticals, transportation of surfactants and lipids in physiological domains, polymer flooding in enhanced oil recovery, gene delivery in biotechnology, and so forth.1–5 Investigations in these areas although cover a wider range, understanding of intricate aspects of the fields remain either weakly explored or unexplored. This is a common feature in scientific research that motivates scientists to advanced studies to gather the knowledge till unknown. The aforementioned problem of polymer (biopolymer)surfactant interaction is a subject of past origin, and accumulated knowledge on the field is available in the literature.6 In recent years, biopolymers like proteins,7 carbohydrate-based polymers,8 and hydrophobically modified polymers9–12 have been considered for interactions with various surfactants and polyelectrolyes,13 employing both classical and modern techniques. A significant interest has been generated by way of fundamental contributions and the scope for practical applications. * To whom correspondence should be addressed. Fax: 91-33-2414-6266. E-mail: [email protected].

The interaction of proteins with oppositely charged surfactants is controlled by both electrostatic and hydrophobic forces, which are sufficiently strong to destroy the tertiary structure of proteins; their iso- and nonisoelectric solution conditions have a significant say on this matter.7,14–18 The investigations are, therefore, often performed at varied pHs and ionic strengths to understand the influence of the above forces of interaction, whose quantification in most cases remains a matter of acknowledged ambiguity. In recent years, we have studied the interaction of surfactants with a variety of proteins and enzymes.14–18 Gelatin (G), derived from denatured collagen, is a random coil polypeptide and a polyampholyte with a nonuniform distribution of at least 18 amino acids, of which glycine (32-35%), proline (11-13%), alanine (10-11%), hydroxyproline (9-10%), glutamic acid (7-8%), arginine (5%), and aspartic acid (4-5%) are the major constituents. Along with the thermoreversible gelling of an aqueous G solution, it acts as an emulsifier, peptizer, stabilizer, film-forming binder, and so forth and thus finds practical applications in pharmaceutical preparation, drug encapsulation, food, gelling agents, photography, and so forth.19,20 In such applications, it is often used in conjunction with one or more surfactants; slow diffusion of G-bound micelles carrying hydrophobic compounds in its core helps the stabilization of dispersions and facilitates gradual mass transfer.21 For photo

10.1021/jp800320a CCC: $40.75  2008 American Chemical Society Published on Web 05/07/2008

6610 J. Phys. Chem. B, Vol. 112, No. 21, 2008 processing purposes, surface tension reduction for multiple layer coatings and wettability of the final coated product, or dispersion of hydrophobic photographic agents like dyes and lubricants, the use of surfactants is required along with gelatin.20 This denatured protein has been physicochemically characterized by its interaction with urea22a and from its adsorption behavior on insoluble stearic acid particles.22b Literature survey reveals that there are exclusive reports on the interaction of anionic surfactant sodium dodecylsulfate (SDS) with G below its isoelectric pH (IEP).23 Above its IEP, a substantial increase in viscosity was observed.24 Clear evidence of the adsorption of micellar aggregates on G at [SDS] , cmc (critical micellar concentration) has been reported from different laboratories.25–43 Complex formation with negatively charged G and polyanions like sodium polystyrenesulfonate (NaPSS) or sodium poly(2acrylamido-2-methylpropanesulfonate) was effective by polarization-induced attraction, as revealed from light scattering studies.44 Interaction of mixed micelles of SDS and a sugarbased nonionic surfactant with G depended both on a critical mole fraction of SDS as well as on the alkyl chain length of the nonionic surfactant.45 However, reports on the interaction of G with nonionic surfactants46 or with cationic surfactants have been strikingly limited.14,15,47,48 The standard free-energy change due to the aggregation of bound cetyltrimethylammonium bromide (CTAB) with G (at pH 5.0, µ ) 0.05, 301 K) was reported to be 18.7 kJ kg-1 of biopolymer, the least among the biopolymer series studied.15 The prolific use of SDS to study its interaction with protein and hence G stemmed from its ability to precipitate proteins from solution under certain pHs and electrolyte solutions; precipitation from a salt-free solution at a pH lower than the isoelectric point for G has been also reported.49 In our earlier studies,14 the possibility of the interaction of G with both SDS and CTAB prompted us to undertake a detailed investigation on the interaction of the protein with the latter at pH 9 (greater than the isoelectric pH 4.84) in a boric acid-borax buffer medium at µ ) 0.005. In addition, the effects on the interaction by varying the temperature and chain length of the cationic surfactants (C12-, C14-, and C18TAB) have been studied in particular. The method of tensiometry was used to understand the interfacial (or surface-active) behavior of the surfactants and G; the other methods, namely, conductometry, turbidimetry, fluorimetry, and isothermal titration calorimetry (ITC), were used for the understanding of the interaction process in the bulk. The cationic surfactants have offered interesting physicochemical behaviors in solution as well as at the interface in the presence of gelatin. The rationalization of the results with pragmatic correlation has been attempted. A comprehensive account of the above is presented in what follows. Experimental Section (A) Materials. AR-grade CTAB of Aldrich (USA, >99%) and AR-grade dodecyltrimethylammonium bromide (DTAB, >99%), tetradecyltrimethylammonium bromide (TTAB, >98%), and octadecyltrimethylammonium bromide (OTAB, >97%) of Fluka (Switzerland) were used. Gelatin (G) (average molecular weight of 38 000 g mol-1 and IEP ) 4.84) was a product of Sigma (U.S.A.) and the sample that we used earlier.14,15 Borax and boric acid were AR-grade products of Merck (India). Pyrene (Aldrich, USA) was a gift sample from the Polymer Science Laboratory of IACS, Kolkata, India. It was purified by vacuum sublimation and then crystallized twice from a 3:1 ethanol-water mixture. The water used in the study was doubly distilled conductivity water of specific conductance 2-4 µS cm-1 at 303

Mitra et al. K. The surfactants produced expected cmc’s, as reported in the literature.50 They did not contain other surface-active impurities as γ (surface tension) versus log [surfactant] plots did not produce a minima (Figure S I, Supporting Information). (B) Methods. All measurements were taken in boric acid-borax buffer solution at pH 9 and ionic strength µ ) 0.005. The biopolymer solution was prepared by dissolving a desired amount of dried G flakes at room temperature into the aqueous buffer solution and allowing the biopolymer to swell for 30 min. It was then heated to 313 K with constant stirring until complete dissolution. The solution was then cooled to room temperature for experimental use. The concentrations of G used were expressed in g % (w/v). Experimentation with [G] higher than 0.2% (the maximum [G] herein used) would be too complex as it leads to increased turbidity in solution, which hinders its detailed study by physical methods. (i) Phase BehaWior. The phase behavior of G was observed visually by varying the concentration from 0.05 to 5% in the studied temperature range of 288 to 320 K. In the actual experiment, 5 mL of G solution at the desired concentration was placed in a stoppered test tube, and the temperature of the sample solution was increased by 2-3° (accurate within (0.1 K) in steps from 288 to 320 K. After equilibration for 30 min, the corresponding phase change at each step was noted. Upon reaching the upper limit, the temperature was then decreased in steps. Each experiment was duplicated to check reproducibility. (ii) Tensiometry. Tensiometric measurements were taken with a calibrated du Nouy tensiometer (Kruss, Germany) by the ring detachment technique. A volume of 10 mL of G solution of the desired concentration (or the buffer solution for dilution experiments) was placed in a thermostatted (accuracy within (0.1 K) double-walled container at the requisite temperature into which a stock ATAB solution of desired concentration (∼10-16 times cmc) was stepwise added with a Hamilton microsyringe as required (allowing 20 min of equilibration time after each addition). The detailed procedure of surface tension measurements has been reported earlier.8,17,51 Duplicate measurements were taken to check reproducibility. The γ values were accurate within (0.1 mN m-1. (iii) Conductometry. The conductivity measurements were performed with a Jenway (UK) conductometer using a cell of unit cell constant. The same procedure of the addition of a stock concentrated ATAB solution (∼10-30 times cmc) to 10 mL of G solution or buffer solution at 303 K (accurate within (0.1 K) as that for tensiometry was followed. After ensuring thorough mixing and temperature equilibration, the specific conductance (κ) was measured. The accuracy of the measurements was within (1%. The measurement details can be found in our earlier reports.8,17,51 (iW) Spectrophotometry. (a) Turbidimetry. An UV-visible spectrophotometer model 1601 of Shimadzu (Japan) operating in the dual beam mode was employed for spectral measurements using a matched pair of quartz cuvettes with a 1 cm path length under thermostatted conditions (303 K) with fluctuations within (0.1 K. The buffer solution was used as the control. The ATAB stock solution (at ∼4-6 times cmc) was then progressively added with a Hamilton microsyringe into the sample cell (consisting of 2.5 mL of G solution of desired strength) as required, and the solution was stirred well for mixing and allowed to equilibrate for 5 min. The spectral measurements were taken in the percent transmittance mode (% T) in the wavelength range of 200-700 nm. The turbidity index (100 % T at 264 nm) was plotted against [ATAB]. At this λ, pure G produced a shoulder in the spectra. The measured values were

Gelatin with Cationic Surfactants ATABs corrected with a blank corresponding to the dilution of protein. The measurement details can be found in an earlier report.8 (b) Fluorimetry. The fluorimetric measurements were taken in a fluorimeter, Fluoromax-P, Horiba Jobin Yvon (U.S.A.), using a 1 cm path length fluorescence quartz cuvette. The experimental procedure was similar to that used for turbidimetry, except that here, both G and the stock CTAB solution contained 1.5 µM pyrene. The procedure details can be found in the literature.52 The emission spectrum of pyrene was scanned from 350 to 450 nm after excitation at 335 nm, allowing an excitation and emission slit width of 5 nm, a wavelength increment of 1.0 nm, and an integration time of 0.10 s. The ratio of the fluorescence intensities at the vibrational peaks of I3 and I1 in the emission spectrum were then plotted against [CTAB].53 (W) Isothermal Titration Calorimetry (ITC). The OMEGA, ITC microcalorimeter of Microcal, Northampton (MA, U.S.A.) was used for thermometric measurements. A concentrated solution of ATAB (DTAB ) 150 mM, TTAB ) 28 mM, CTAB ) 18 mM, and OTAB ) 4 mM) in the 350 µL microsyringe was added for an injection duration of 30 s to 1.325 mL of G solution (in the calorimeter cell) at equal intervals of 240 s in multiple steps (32-42 additions) under constant stirring (350 rpm) conditions. An identical G solution (1.645 mL) was taken in the reference cell. The heat released at each step of interaction of the surfactant with G was recorded, and the enthalpy per mole of ATAB added was calculated with the ITC Microcal Origin 2.9 software.8,14,16,17,51 The experiment of the dilution of buffered ATAB was also performed with the same injection matrix as that of the interaction experiment, placing the aqueous buffer solution in the reference and calorimeter cell. The enthalpy of the dilution (∆Hd) or interaction (∆Hi) process was then plotted against [ATAB]. Each run was duplicated to check reproducibility. Water was circulated within the calorimeter by a NESLAB RTE100 bath at a temperature lower within 5° of the experiment temperature. The temperature in the cell compartment of the calorimeter was automatically scanned up to the desired temperature of the measurement and adjusted with an accuracy of (0.01 K. Results (A) Pure G. (i) Phase BehaWior. The temperature influenced phase behaviors of G solution are presented in Table S I (vide. Supporting Information). A 5% G solution formed gel at and below 300 K. The gelation temperature diminished with decreasing [G]. The 1% G solution remained in the liquid form throughout the studied temperature, except at 288 K where it was a viscous liquid. However, [G] < 1% remained in the liquid state. As per ref 38, photographic-grade G gelates at 308 K (concentration not mentioned). The gelation temperature of 5% G within 298-303 K was also reported.21 In this study, we have used 0.005-0.2% [G] in the temperature range of 293-313 K. Thus, the results were not affected by the internal consistencies of the biopolymer solution. (ii) Interfacial Adsorption BehaWior. The buffered solution of G has shown surface activity. It has been documented in Table S II (A) (Supporting Information). According to earlier reports,36a the γ of 7 wt % G at 313 K was 38.8 mN m-1, which in the presence of 1 M NaCl was 35.8 mN m-1. Proteins are normally surface-active and may suffer surface denaturation. The denatured conformation of gelatin exposes its peripheral ionic sites, easing solubilization of the protein in the buffer through hydration. The surface activity of G diminished with increasing temperature [Table S II (B), Supporting Information]. The temperature decreased the hydration of gelatin at the

J. Phys. Chem. B, Vol. 112, No. 21, 2008 6611 interface. The protein thus became desorbed from the surface, as reported earlier.33 (B) Micellization of Pure ATAB in pH 9 Buffer at 303 K. (i) Tensiometry. The tensiometric isotherms of pure CTAB in buffer solution evidenced an initial mild surface activity with a sparsely populated interface with CTAB monomers. It was then followed by an enhanced activity with a sharp decline in γ up to completion of monolayer formation at γcmc ) 33.2 mN m-1, and, thereafter, it remained unchanged. The threshold surfactant concentration at which γ leveled off was the cmc of CTAB. The tensiometric way of determining the cmc’s of ATABs is illustrated in Figure S I (Supporting Information), focusing on DTAB and TTAB in particular. Such results are quite often found in the literature.50 The alkyl chain length of the ATABs controlled the self-aggregation process. The cmc increased with decreasing chain length. A nearly constant γcmc was registered for the ATABs, although their chain length varied. The relative Gibbs surface excess of the saturated CTAB monolayer at the air/solution interface (Γmax) was calculated from the slope of the linear profile of the tensiometric isotherm up to cmc according to the well-used Gibbs adsorption equation.8,17 The area of exclusion per monomer (Amin) and the standard Gibbs free energy of interfacial adsorption (∆G°ad) were calculated following the procedures and equations used in earlier studies (see footnotes f and h of Table S III in the Supporting Information for relevant equations).17 Greater tilts of the longer ATAB molecules described greater Amin.50a It was found that the interfacial adsorption of ATABs was more spontaneous than the micellization process. (ii) Conductometry. While tensiometry is concerned with the amphiphile behavior at the interface, conductometry responds to bulk phenomena. All of the ATABs produced a linear dependence between κ and [ATAB], with breaks at cmc to mark the onset of micellization. The ratios of the slopes for the post cmc and pre cmc profiles, S2 and S1, respectively, were considered to evaluate the counter Br- condensation (f) to the micellar surface using Evan’s equation (Figure S I, inset in Supporting Information for illustrations).54a Such plots are frequently found in the literature.8,17,50 The standard Gibbs free energy of micellization of pure ATAB was calculated as earlier.17,50,51 The fRTlnXcmc term (Xcmc ) cmc of pure ATAB on the mole fraction scale; T ) absolute temperature) on the whole contributes to the electrical free energy of micellization.54b The standard state is the hypothetical state of the unit mole fraction. The results have evidenced that the spontaneity of selfaggregation increased with increasing ATAB chain length (see footnote g of Table S III in the Supporting Information for relevant equations). (iii) Microcalorimetry. The ITC enthalpograms are well documented in the literature;8,14,16,17,50,51 results on DTAB are only depicted (see Figure S I, inset, in the Supporting Information). The cmc and enthalpy of the micellization process, ∆Hm, determination rationales are indicated on the curve.55 The ITC cmc’s correlate with those of other methods and reports.50 The exothermicity of the process has increased with increasing hydrophobicity of the ATAB members due to increased intermolecular association. The calorimetric ∆Hm of CTAB produced a constant 1 atm pressure specific heat capacity (∆Cpm) of -510 ( 50 J K-1 mol-1 in the buffer medium as compared to -520 ( 20 J K-1mol-1 in the non-buffered medium.8 It is worthwhile to report the heat capacity obtained by the van’t Hoff procedure -1 mol-1, much greater than (∆CpVH m ). It was -3230 ( 30 J K the direct method of calorimetry. Such discrepancies between

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Figure 1. The γ versus [CTAB] plots in the absence and presence of G in a buffer solution at pH 9. The solid symbol represents the profile without G. Error bars are shown in the profiles. The open symbol represents the course in the presence of 0.2% G. T1, T2(cac), T3, CT, CM, CB, and T4 (cmc2) marked in the profile are discussed in the text. The identification of the states Ia, PA Ib (process A corresponding to Scheme 1b), PA′ Ib, PB Ic, and so forth are in reference to Scheme 1, discussed subsequently.

the two methods were reported by us in the past.50b,56 The calorimetry evaluates the integral enthalpy (enthalpy for all involved solution processes in addition to micellization), but the van’t Hoff procedure is a differential method related to amphiphile association (including counterion binding for ionics) in solution. The associated errors in the determination of ∆HVH m from ∆G°m values at different temperatures also contribute to the difference57 (see Table S III in the Supporting Information for interfacial/bulk parameters for ATAB micellization). (C) G-CTAB Interaction. (i) Effect of [G] Variation. The tensiometric profiles for the interaction of CTAB with 0.005-0.2% G have evidenced various transitions in them. Illustrations for 0.2% G with error bars are shown in Figure 1. At pH 9, the acidic residues of G, namely, aspartic acid (pKa ) 4.5), glutamic acid (pKa ) 4.5), and histidine (pKa ) 6-7.5), were all negatively charged, and CTA+ ions associate, forming surface-active species that effectively decreased γ to a point T1 that arose by way of electrostatically driven cooperativity for strongly interacting systems.58–60 Further addition of CTAB arrested the decline of γ with the appearance of a halt in the tensiometric profile in the T1-T2 region. For a strong polymer-surfactant interaction, the interfacial process can have control over the bulk with a substantial change in activity of the amphiphile.25,61 At T2 (0.05 mM for 0.2% G, which is the cac), small amphiphile aggregates started to form and became associated with the polymer chain like “beads” in a necklace. For the 0.5% G-CTAB system, cac was observed at a much higher concentration of 2 mM; however, specific binding was observed up to a concentration of 0.2 mM.48 The dependence of T2 on [G] was not as strong as that reported elsewhere.17,21,38,60a Prior to the formation of T2, the T1-T2 region narrowed down with decreasing [G] and virtually merged into a single point at [G] ) 0.05%. Thereafter, γ of the system increased, as evidenced in the region between T2 and T3. T3 enhanced with increasing [G]. In very dilute solutions of G, the transition points from T1 to T3 were too mild to be monitored by tensiometry. Between T3 and CT (turbidity visibility point), a second plateau at γ ∼ 36.0 mN m-1 arose at all [G]. As will be evidenced later from spectroscopic results, the coacervation phenomenon was initiated at T3 and continued until CT; the formation of aggregates with the distinct appearance of turbidity at higher [G] was visible. With further CTAB addition, turbidity increased

Mitra et al. and maximized at CM forming a hump. At lower [G] and at higher temperature (discussed later), the hump formation after CT was not apparent. The rise in height virtually remained invariant with [G]; for the NaPSS-CTAB system, the height was found to have inverse dependence on [polymer].62 It may be mentioned here that in the NaPSS-CnTAB (n ) 10, 12, 14, 16) system, both the polyelectrolyte and the surfactant were present at the air/solution interface at concentrations much lower than that for T1 to above the formation of the free micelle.62 This increase in γ was earlier considered to be the consequence of an equilibrium between the surface and a dilute solution phase following precipitation of the polymer-surfactant aggregate complex.59 The increased presence of CTAB in the system caused precipitation of the complex (at CB, turbidity starts dissolving, as observed visually) that remained dispersed in solution. Thereafter, free CTA+ ions started accumulating at the interface. Both γ and turbidity decreased up to T4 to mark the onset of free CTAB micelles in solution, designated as cmc2 (extended cmc), beyond which γ hardly changed, forming a third plateau. T4 enhanced with increasing [G]. Similar to the present findings, an extended cmc of SDS was also reported by way of interaction with G.21,38 The T1, T2, T3, T4, and the turbidity-related values CT and CM are presented in Table 1a, along with results by other methods, to be discussed subsequently. A nearly constant γT4 for all [G] meant comparable 4 ) for the interfacial composition. The Gibbs surface excess (ΓTmax free micelle formation of CTAB between CM and T4 in the presence of G, the area of exclusion per CTAB headgroup at 4 ), and the free energy of adsorption the air/solution interface (ATmin ° ° (∆Gad,T4) using ∆Gm,T4 values were obtained as before (see footnotes f and h of Table S III in the Supporting Information for relevant equations).17,50,51 The required πT4 values were calculated from the differences between γG [Table S II (A), Supporting Information] and γT4. An enhancement of the interfacial adsorption of CTAB with decreasing [G] was obtained. Thus, G produced an unfavorable environment for CTAB adsorption probably by the effect of its self-surface activity (see Table S IV (a) in Supporting Information for interfacial adsorption parameters). In the presence of 0.005-0.2% G, the conductometric plots of CTAB have shown several distinct differences. Figure 2 illustrates the interaction with 0.1% G. Three distinct regions, which fairly corresponded to T3, CT, and T4 of the tensiometry profiles, are marked in the plot. The transitions are also found in the derivative plots of conductance. Initially, the conductance increased with CTAB addition nearly linearly until the point T3, where coacervation started to form in solution. Between the region T3 and CT, κ increased with a greater slope, and after CT, the slope was lower up to T4. Further lowering of the slope was observed in the post T4 region. In the T3-CT region, visible aggregates of the CTAB complex were noticed. Mild breaks at CT were observed for the lower two concentrations of G. The various transition points agreed fairly well with other methods. A low counterion binding (fCT) of the G-bound smaller micelles (compared to 0.68 for pure CTAB micellar aggregates) was in line with earlier findings.17 The increase in fCT with increasing [G] was noticed. The associated free-energy change at CT (∆G°CT) was enhanced with increasing [G]; higher [G] facilitated greater interaction and easier coacervation in solution. The conductance decreased in the post cmc region (T4) like any other ionic self-aggregating system. The break was prominent; the condensation of counter Br- from the solution into the Stern layer of the surfactant aggregates was evident. The fT4 decreased with increasing [G]; the influence of the polymer matrix

Gelatin with Cationic Surfactants ATABs

J. Phys. Chem. B, Vol. 112, No. 21, 2008 6613

TABLE 1: The T1, T2 (cac), T3, CT, CM, and T4 (cmc2) (all in mM) for the Interaction of G-ATAB in pH 9 Buffer Solution at 303 K; (a) G-CTAB System at Varying [G], (b) 0.05% G-ATAB System at Varying ATAB tensiometry system

T1

T2

T3[CT]

conductometry T4[CM]

T3[CT]

microcalorimetry T3[CT]

T4

T4

turbidimetry (fluorimetry)a T3[CT]

T4

(a) b

0.005% 0.025%b 0.05% 0.1% 0.03 0.2% 0.02 OTAB CTAB TTABb DTABb

0.16 [0.20] 0.83 [-] 0.06 [0.21] 0.87 [0.32] 0.03 0.14 [0.32] 1.04 [0.38] 0.04 0.17 [0.37] 1.58 [0.56] 0.05 0.20 [0.56] 2.08 [0.80]

0.009 0.016 0.03 [0.13] 0.60 [0.16] 0.03 0.14 [0.32] 1.04 [0.38] 0.08 [1.88] - [-] 1.48 [12.4] - [-]

- [-] 0.10 [-] 0.14 [0.35] 0.15 [0.49] 0.16 [0.62]

0.85 0.99 1.22 1.35 1.70 (b)

0.03 [0.14] 0.52 0.14 [0.35] 1.22 4.23 14.2

0.10 [0.17] 0.11 [0.28] 0.15 [0.32] 0.17 [0.44] 0.19 [0.58]

0.87 1.05 1.08 1.34 1.66

(not obs)[0.12]c 0.64 0.15 [0.32] 1.08 3.69 14.0

0.10 [0.25] 0.11 [0.28] 0.14(0.12) [0.32(0.35)] 0.17 [0.34] 0.20(0.18) [0.58 (0.60)] 0.06 [0.16] 0.14 [0.32] 0.22 [1.79] 9.18 [12.1]

0.85 0.90 1.14(1.02) 1.50 1.92(1.71) 0.55 1.14 4.39 14.8

a Fluorimetry derived CB values were 0.49 and 1.08 for 0.05 and 0.2% G, respectively. b For 0.005 and 0.025% G, the transition points T1, T2, and T3 were too mild to be identified. The same is true for TTAB and DTAB. c The CB for the OTAB system is at 0.29 mM.

SCHEME 1: Schematic Representation of G-CTAB (or OTAB) Interaction with Reference to the Tensiometric Observations Depicted in Figure 1a

a: G at the interface (GI) and in the bulk (GB). b: Initial formation of GSIn mainly at the interface [process A, (PA)] with trace GSBn in the bulk B [process A′, (PA′)]. c: Bead-like small micelles attached to G forming GSm [process B, (PB)] with a small amount of GSIn at the interface. d: More B C GSm formed [PB] and associated to yield coacervate GSm that phase separates out from solution [process D, (PD)]. CTAB monomers occupy the interface [process C, (PC)]. Coacervates disintegrate in the post CM region. e: Formation of free and bigger micelles in solution along with the necklace bead products GSm(CTAB)MS [process E, (PE)]. CTAB monomers saturate the air/buffer interface [PC] (with trace GSIn at the interface). a

environment on the counterion affinity of the otherwise free CTAB micelles was envisaged. The first and second breaks T1 and T2 observed in tensiometry as well as the turbidity-related state CM were absent in conductometry; the absence of the cacforming stage (T2) is often found in polymer-surfactant interacting systems.17 The associated free-energy change at T4 ° (∆Gm,T 4) was found to decrease with increasing [G]. The magnitude of the free-energy results in the table suggested that coacervate formation was a more favorable process than free micelle formation in the biopolymer environment [see Table S IV (a) in the Supporting Information for thermodynamic parameters].

In solution, the turbidity arose by the formation of visible aggregates in solution. With reference to the inset of Figure 3, for 0.2% G, an inflection occurred at T3, as also observed in the methods of tensiometry and conductometry. The turbidity then increased up to CT and declined from CB up to the point T4, forming a distinct break; the decrease thereafter was mild. The extent of turbidity was enhanced with increasing [G]. It is well accepted that the ratio of the third to the first vibrational peaks of pyrene fluorescence (I3/I1) is a measure of the polarity of its environment.63 The strong hydrophobicity of the pyrene molecules makes them highly distributed in a nonpolar environment such as a micellar interior, with the

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Figure 2. Conductometric plots for CTAB without and with G at 303 K in buffer solution at pH 9. Interaction profiles with 0 and 0.1% G. In the 0.1% G profile, the breaks T3, CT, and T4 are indicated. Plots of the derivatives of κ versus [CTAB] are also displayed in the figure (y-axis on RHS).

Figure 3. Fluorimetric and turbidimetric representations for G-CTAB systems at 303 K in buffer solution at pH 9. Main plot: The (I3/I1) versus [CTAB] profile for interaction at 0.05 and 0.2% G. The T3, CT, CB, and T4 break points are marked on the 0.2% G profile. The lower horizontal broken lines represent (I3/I1) for 0.05 and 0.2% G solutions without CTAB. Inset: Turbidimetric plot for 0.005, 0.025, and 0.2% G in buffer solution. The T3, CT, CB, and T4 points are marked on the 0.2% G profile.

consequence of a large increase in the polarity index, (I3/I1). The (I3/I1) versus [CTAB] profile for pure CTAB has been found to be sigmoidal in nature, with a transition at 0.82 mM, which is the cmc of the amphiphile (plot not shown) in terms of Sigmoidal Boltzmann fitting.50a The polarity index was 0.64 in aqueous buffer of pH 9. The index values were 0.67 and 0.69 in 0.05 and 0.2% G, respectively (Figure 3, main plot). Thus, pyrene remained in the domains of the hydrophobic sites of G; 0.05% G had a larger exposure to water than 0.2%.53 The index increased upon addition of CTAB into the G solution up to T3 before forming a plateau similar to that reported earlier.53 The formation of the plateau region was more distinct in 0.2% G than that in 0.05% G; the hydrophobicity of the region increased with increasing [G]. The absence of T2 (cac) by spectrophotometry was similar to that observed earlier.17,38 The fluorescence measurements in the turbid region CT were a bit erratic. A fall in the index values occurred in the turbid zone from CT to CB. It is only in fluorimetry where CB could be distinctly identified from the plots. Past the point CB, the turbidity diminished with increased (I3/I1), which maximized at T4 and subsequently remained constant at (I3/I1) ) 0.89. The transition

Mitra et al.

Figure 4. Enthalpograms for dilution of CTAB in buffer solution (pH9) and its interaction with varying percentages of G at 303K. Main plot: Profiles in 0, 0.025, and 0.1% G. Inset: Locations of the T3, CT, and T4 states and the corresponding ∆HT3, ∆HCT, and ∆HT4 evaluation rationale from the enthalpogram are illustrated.

points obtained from spectrophotometric methods agreed fairly well with other methods. In the present context, a comparative discussion on the G-CTAB interaction at pH 7 by Saxena et al.48 would be worthwhile. From the dynamic light scattering measurements, they have observed that the hydrodynamic radius (Rh) of G increased in the presence of CTAB by way of expansion of the biopolymer in solution. At pH 7, the binding of CTAB (effectively CTA+ ions) to G should reduce the effective negative charge on the protein, and hence, chain expansion was not expected. In our view, the increased Rh was due to growth of coacervate by aggregation (cf. process D presented in the Discussion section), clearly observed from spectrophotometric measurements. Besides, we have observed the appearance of the enhanced turbidity of G at concentrations above 0.2% at 303 K, which was expected to be more at 298 K. They used 0.5% G in their study. It was quite possible that aggregates of G and the G-CTAB complex produced increased Rh; expansion of the protein chain was not the reason for the results. The identification and enthalpic quantification of several interaction processes were achieved by the ITC method, as reported earlier.8,14,16,17,64,65 The enthalpograms in microcalorimetry have evidenced the initial rise and subsequent decline after a peak (at T3) to more or less leveling off at higher [CTAB] in the presence of G (Figure 4, main plot). The enthalpic activities of G at a higher concentration were more than those at the lower one. The realized curves had three distinct sections: an initial endothermic rise (∆HT3) followed by an exothermic fall in two stages (∆HCT and ∆HT4) (Figure 4, inset). The related endothermicity to T3 (∆HT3) became more pronounced with increasing [G]. Addition of more CTAB sharply decreased the interactional enthalpy from ∼CT with a change in slope at [CTAB] ) T4. The exothermicity of the second process (∆HCT) also increased with increasing [G]. A further decline in enthalpy was observed in the post T4 region, and the curve flattened out subsequently with minor changes. As mentioned earlier, T4 was the point of extended cmc; the related enthalpy (∆HT4) has been found to decrease with increasing [G] (Table 2a). The binding of CTAB to G at 0.03-0.05 mM (T2) identified in tensiometry was not recognized in calorimetry for sensitivity reasons. (ii) Effect of Temperature Variation. Temperature has an influencing effect on the interaction with oppositely charged polymer surfactant systems, as is expected for systems that are partly driven by hydrophobicity.66 The tensiometric profiles of 0.05% G with CTAB addition within a temperature range of 293-313 K are depicted in the

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J. Phys. Chem. B, Vol. 112, No. 21, 2008 6615

TABLE 2: Determination of the Interaction Enthalpies of the G-ATAB System in pH 9 Buffer Solution by the Microcalorimetric Method for (a) the G-CTAB System at Varying [G], (b) the 0.05% G-CTAB Interaction at Varying Temperatures, and (c) the 0.05% G Interaction with Varying ATAB -∆Hi/kJ mol

-1a

system

-∆HT3

-∆HCT

-∆HT4

3.80 5.66 5.68 6.63 7.08

9.24 8.24 7.75 7.56 7.40

3.42 5.26 5.68 7.91 12.1

5.71 7.47 7.75 8.08 8.84

(a) 0.005% 0.025% 0.05% 0.1% 0.2%

-1.90 -3.07 -3.27 -3.97 -4.12 (b)

293 298 303 308 313

-5.14 -5.06 -3.27 -2.90 -2.79 (c)

OTAB CTAB TTAB DTAB a

-, (∆HCB ) 9.57) -3.27 -

2.25 5.68 -

7.75 3.39 4.81

SD for the ∆Hi’s is (2%.

Figure 5. Temperature-dependent tensiometric and thermometric plots for CTAB in 0.05% G in buffer solution at pH 9. Main plot: Tensiometric results at 293, 298, 308, and 313 K. The features are the same as those depicted in Figure 1 for 0.2% G. Inset: Enthalpograms at 293, 303, and 313 K for the 0.05% G-CTAB interaction.

main plot of Figure 5; the features are comparable with those of Figure 1. The breaks at T1 and T2 at 293 and 298 K in 0.05% G solution were absent; they were comparable with the results with 0.005 and 0.025% G at 303 K. The temperature produced a mild effect on T2. At higher temperature, morphological changes in G exposed more ionic sites to induce a greater interaction. The temperature effect has evidenced a variation in γ in the range of 35.5-37.6 mN m-1 at the surface saturation level (second plateau), in agreement with an earlier report.8 At the two lower temperatures, the solution remained turbid, which decreased with increasing temperature.66 Contrary to our observation, temperature variations of 5 and 40 °C on NaPSS-DTAB produced no perceptible change in the phase behavior of the system.67 T4 increased with temperature since the micellization process was exothermic. The various transition points obtained from tensiometry and microcalorimetry are presented in Table 3. The surface excess of CTAB monomers

TABLE 3: The Transition Points [T1, T2(cac), T3, T4(cmc2), CT, and CM (expressed in mM)] in the Interaction of CTAB with 0.05% G in pH 9 Buffer at Varying Temperatures tensiometry T/K

T1

293 298 303 308 0.02 313 0.02

T2

T3[CT]

microcalorimetry T4[CM]

0.02 [0.20] 0.71 [0.22] 0.11 [0.22] 0.93 [0.25] 0.03 0.14 [0.32] 1.04 [0.38] 0.03 0.16 [0.45] 1.26 [-] 0.04 0.19 [0.49] 1.57 [-]

T3[CT]

T4

0.13 [0.29] 0.14 [0.31] 0.15 [0.32] 0.16 [0.50] 0.18 [0.59]

0.76 0.93 1.08 1.25 1.60

was reduced with increasing temperature, an observation similar to that for pure CTAB.8 However, γT4 followed an opposite trend compared with the effect of temperature on pure G and CTAB [see Table S IV (c) in the Supporting Information for interfacial adsorption parameters]. Temperature has a delaying effect on coacervation (T3). The enthalpograms of the G-CTAB interaction have followed similar patterns at all of the studied temperatures (Figure 5, inset). The cmc of pure CTAB has a minimum at 303 K, whereas the extended cmc (T4) observed from calorimetry has increased with increasing temperature. The environmental conditions in the biopolymer solution were different from those in aqueous medium without G. The endothermicity of the initial process lessened with the rise in temperature, and the exothermicities of the other two processes of coacervation and extended micellization increased with increasing temperature (Table 2b). The interplay of the hydrophobic interaction in the thermodynamics of the process was envisaged. (iii) Effect of Alkyl Chain Length Variation. The tensiometric profile with transitions for OTAB is presented in the main plot of Figure 6. The profiles of TTAB and DTAB are flat to yield broad plateau regions up to the cmc (Figure 6, inset). Thus, identification of different stages was not possible. The observed transition points obtained for OTAB by different methods are presented in Table 1b. By analogy with the tensiometric results of NaPSS-CnTAB (n ) 10, 12)62 and polyethyleneimine-SDS at pH 10, such behaviors may have arisen for lower ATAB homologues if the adsorption of the polymer/surfactant complexes at the interface were of multilayer character. Neutron reflectivity measurements have clearly indicated the presence of a thick adsorption layer (or layers) at the interface for lower chain length surfactants and a thin layer for higher chain lengths.62 Flattened tensiometric profiles have been also found

Figure 6. Tensiometric plots for pure ATABs in 0 and 0.05% G at 303 K in buffer solution at pH 9. Main plot: For OTAB in 0 and 0.05% G. The features of the profile are the same as those presented in Figure 1 for 0.2% G. Inset: Similar plots for DTAB and TTAB in 0 and 0.05% G. The observed features were mild.

6616 J. Phys. Chem. B, Vol. 112, No. 21, 2008

Figure 7. Turbidimetric and microcalorimetric representations for the ATAB interaction with 0.05% G at 303 K in buffer solution of pH 9. Main plot: Turbidity profiles for DTAB and TTAB. General features of the profile are comparable to that described in Figure 3 (inset) for 0.2% G. Inset: Enthalpograms for OTAB in 0 and 0.05% G solution. The CT,CB, and T4 states are indicated in the plot.

to be produced by the SDS/poly(vinylpyridinium chloride)/NaCl (0.1 M) system,60a by sodium tetradecylsulfate/polydimethyldiallylammonium chloride/0.1 M NaCl,60b and by the 7 wt % G-SDS system.36a In the latter case, a dilational rheological study has evidenced multiple adsorption features at the air/ solution interface. Interfacial parameters were not obtainable for the lower ATAB homologues because of the absence of the transition points between T1 and T4 in tensiometry. Although feeble, the CT and CB states were visually observed for DTAB and TTAB. In the T4 region, population of OTAB in the presence of G was slightly lower than that for its monolayer in the absence of G. On a comparative basis, the surface density of OTAB was ∼50% lower than that of CTAB in 0.05% G [see Table S IV (b) in Supporting Information for interfacial adsorption parameters]. The conductance behavior of the other ATABs (TTAB and DTAB) in the 0.05% G solution was quite different from that of CTAB and OTAB that produced distinctive breaks at T3, CT, and T4 (cf. Figure 2 for CTAB). For the lower two homologues DTAB and TTAB, there was only one break in each case, with minor differences between the conductance profile with and without G in solution (plots not illustrated). The complex of G with OTAB aggregates favorably condensed more Br- to yield higher fCT values. The fCT registered by OTAB was ∼1.5 times greater than that of CTAB. However, interaction with the lower two homologues resulted in no perceptible change in the counterion binding; probably the extent of interaction was too small to register a change. The single breaks in the plots fairly corresponded to T4 by turbidimetry and were mildly greater than the pure cmc’s. Interestingly, the micellization of ATABs in buffer and in the presence of 0.05% G have ended ° up with comparable ∆G°m and ∆Gm,T 4 values [see Table S IV (b) in Supporting Information for thermodynamic parameters]. The maxima in the turbidity plots for the ATABs in 0.05% G followed a decreasing trend with decreasing alkyl chain length. The hydrophobicity of the alkyl chain played a controlling role in precipitation.69 Turbidimetric profiles for DTAB and TTAB are only depicted in the main plot of Figure 7. There, T3 corresponded to the onset of phase separation of the complex in solution. With more ATAB addition, the complex separated out as visible aggregates. Excess ATAB initiated a turbidity decrease at CB with a clear solution at T4. The observed transitions (T3, CT, and T4) fairly agreed with other methods. While tensiometry and conductometry produced similar profiles for OTAB and CTAB, their ITC enthalpograms were

Mitra et al. dissimilar. The method failed to register T3 at such a low [OTAB] (Figure 7, inset), as discussed earlier for micellization of surfactants having low cmc.70 In the beginning, the interaction enthalpogram of OTAB produced two successive peaks; the first matched with CT, and the second corresponded to CB. Beyond T4, the enthalpy decreased sharply. Because of solubility restrictions, the experiment could not be performed with higher concentrations, and hence, the ∆HT4 estimation for OTAB remained indecisive. The two peaks merged into one for CTAB and became very minor or absent for TTAB and DTAB (not illustrated). Although OTAB and CTAB demonstrated extended cmc (T4), DTAB and TTAB in 0.05% buffered G solution produced cmc breaks comparable with their aqueous cmc’s. The enthalpies for the endothermic rises (for OTAB and CTAB) and the exothermic declines for the ATABs at 0.05% G are presented in Table 2c. For DTAB and TTAB, only ∆HT4 was obtained. ∆HCT for OTAB was greater than that of CTAB. The process significantly changed with the reduction of two methylene (-CH2) groups in the alkyl chain. Higher homologues of long-chain alkylammonium chlorides have been also reported to favorably bind with DNA.57 In the case of negatively charged G, ATAB binding was appreciably dependent on the alkyl chain length. Discussion The discussion presented below is with reference to the interaction model shown in Scheme 1 and the identified zones in Figure 1. At the temperature and concentration used in this study, gelatin solution was easy flowing and nonviscous. The protein was fairly surface-active (Scheme 1a) that increased with [G] and decreased with temperature. The presence of the hydrophobic domains of gelatin at the interface lowered the surface tension, and its desorption at higher temperature increased γ. The interaction features have six distinct regions. 1. Pre T1 (Scheme 1 b): In the low [surfactant] regime, monomers (S) preferentially adsorb onto the oppositely charged peripheral ionic sites of G at the interface (GI), resulting in formation of the interfacially active G-CTAB complex (GSIn) to effectively decrease γ by the process

GI + nS ⇒ GSIn

(Process

A or PA)

where n number of S’s became attached to different anionic centers of GI. This process continued up to a certain point of addition, say T1. Depletion of solvation of G by the formation of the complex resulted in an increase in hydrophobicity. This has been supported from an initial steep rise in the (I3/I1) value by fluorimetry. However, the sparse binding of both OTAB and CTAB monomers to G made the conductance method insensitive to register T1. The process was more entropy-driven than enthalpy-driven and was insensitive to calorimetry.71 The solution was also turbidimetrically transparent. 2. T1-T2 (Scheme 1 b): Further addition of CTAB caused the CTA+ species to favorably combine with G in the bulk (GB)

GB + nS ⇒ GSBn (surface inactive complex) (Process

A′ or PA′)

thereby arresting the decline of γ. The domain of this initially formed plateau became inflated with increasing [G] due to the formation of enhanced GSBn . 3. T2-T3 (Scheme 1 c): The favorable interaction of CTAB with G induced an early aggregation of the amphiphile at T2, which is called the cac point. Beyond T2 until T3, desorption of

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J. Phys. Chem. B, Vol. 112, No. 21, 2008 6617

GSIn from the interface to the bulk occurred with the simultaneous transformation of it to a polyelectrolye-type complex, GSBm, via hydrophobic and electrostatic interactions with CTAB aggregates according to the scheme B GSIn + CTAB aggregates ⇒ GSm

(Process

B or PB)

Here, Sm is the amphiphile aggregate formed at a concentration greater than cac, making γ increase up to T3. Enhancement in T3 with increasing [G] was due to a greater interaction of G resulting in an increased [GSIn] conversion to [GSBm]. For 0.005 and 0.025% G, the transitions from T1 to T3 were too small to detect because of negligible [GSIn] formed at the interface. However, methods other than tensiometry failed to monitor this event. Hydrophobic association of CTAB micellar aggregates with GSIn was also identified from an increase in the polarity index ratio (I3/I1) of pyrene up to T3. However, the pyrene environment was unable to detect a difference in the hydrophobic nature between GSIn and GSBm. Electrostatic cooperative binding was an overall endothermic process (∆HT3), as observed from microcalorimetry, and the endothermicity increased with increasing [G], registering increased amphiphile binding. 4. T3-CT (Scheme 1 d): At this stage, replenishment of the relieved interface with CTAB species occurred following the process

I + xCTAB ⇒ I-(CTAB)x

(Process

C or PC)

GSBm

Also, the complex started to form coacervates (evidenced from spectrophotometry) following the transformation B B yGSm ⇒ (GSm )y or GSmC

(Process

D or PD)

where GSCm was a colloidal dispersion having the tendency to separate out from the solution but, to start with, remained undetectable to naked eye. Beyond T3, the rates of the three processes (B, C, and D) maintained a balancing compromise between them to control the nature of rise and fall in γ in this region. The γ remained to be ∼36.0 mN m-1, justifying surface saturation with CTAB monolayers only with a mild fall at higher concentration; the slight variation of it for [G] g 0.05% was caused by the presence of adsorbed GSIn at the interface. Thus, the consecutive B C transformation process GSnI ⇒ GSm ⇒ GSm continued with incoming CTAB in the system until CT. Manifestation of coacervation (process D) was clearly evidenced from a steep rise in turbidimetry at T3. However, a small halt in the fluorimetric profile between T3 and CT meant that pyrene initially lodged within the hydrophobic moiety of GSBm failed to detect the coacervation initially. The binding process in this regime that accounted for a slope enhancement in the conductometric profile can be explained by considering three possibilities, (a) depletion of the counter H+ ion from the G chain, (b) dissociation of a part of the bound Br- ions from the CTAB aggregates by way of morphological changes in this region, or (c) conduction by the charged colloidal polyelectrolyte-type complexes formed in the process. More than one process could have contributed to the results. The above explanation is only tentative; for a conclusive elucidation, further study is warranted. 5. CT-T4 (Scheme 1 d): The residual amount of GSIn still remaining at the interface associated with CTAB aggregates to form more GSBm with a further increase in γ up to CM. Thereafter, further incorporation of CTAB disintegrated the aggregates. It tended to separate out from solution. A decrease in the conductance after CT was via the loss of formed aggregates from solution into the coacervate phase. The increase in fCTwith increasing [G] was a consequence of this altered morphology

of the G-bound amphiphile aggregates. The overall process of coacervation was an exothermic process. Destabilization of the coacervates with a sharp decline in the (I3/I1) profile from CT to CB was due to disintegration of coacervates lodging pyrene. Pyrene, upon being exposed to a more polar environment register a decreased index value. The influence of turbidity on the spectral manifestation of the system also affected the polarity index. Increased population of CTAB at the interface made γ drop in the post CM region following process C. 6. Post T4 (Scheme 1 e): Interfacial saturation registered a constant γ at T4 to mark the onset of formation of cmc2 (extended cmc). T4 increased with [G] due to an increased number of sites for thermodynamic association of the biopolymer with CTAB aggregates. Counter Br- condensation in the double layer of free micelles registered a decline in conductance in the post T4 region. We anticipated a network structure of the micelle-decorated necklace bead ensembles of G (as also modeled by Saxena et al.48), which remained solubilized in the free micellar solution (denoted as “MS”) following the scheme72,73 C GSm + free CTAB micelle S GSm(CTAB)MS (soluble) +

free CTAB micelle

(Process

E or PE)

Fluorimetry also registered micellization through a rise in the (I3/I1) ratio in the post CB region. An unaltered polarity-related environmental condition was indicated with a probability of G-bound amphiphile aggregates in association with CTAB micelles, that is, GSm(CTAB)MS. The same maximum (I3/I1) value of 0.89 with both 0.05 and 0.2% G justified the view. 4 with increasing [G] meant an associated lower Lowering of ΓTmax surface density of CTAB in the post CM region to reach T4; this might be due to interfacial occupancy of a part of G or its T4 complex GSIn to register higher Amin at the interface. The interface was effectively independent of [G], rich in pure CTAB, and with a minor contribution from the G-CTAB complex. There were similar reports from other studies.8,59,74 The free micelle formation was exothermic (∆HT4), as found from calorimetry. The thermal condition of the protein solution had influenced its interaction with CTAB; higher temperature augmented a greater interaction with increased T2. Until a sizable proportion of surface-active GSIn was transformed into surface-inactive GSBm, the appearance of T3 was delayed. The coacervation process was enhanced with temperature. Temperature favored the C dissolution of the complex (coacervate) GSm in the micellar environment following process E. The complex was surrounded by solvent molecules whose disruption at higher temperature augmented effective disintegration of GSCm. After complete dissolution of the aggregated species, the morphology of the GSm(CTAB)MS formed depended on the thermal state of the solution, as per the expectation on other physicochemical conditions. This warrants further examination. The hydrophobic chain length of the ATAB homologues had a say in their interaction potential with G in buffered conditions. OTAB and CTAB produced similar features and were fairly interactive. The lower two homologues, DTAB and TTAB were poor in this respect. It was possible that the surface interactions masked the changes occurring in the bulk, as observed earlier.25,61 Low turbidity indexes suggested a low degree of coacervation for both of them. Nearly equal γT4 values for pure and G-interacted DTAB or TTAB might be due to a too insignificant contribution of G or its complex at the interface. The tensiometry, conductometry, and microcalorimetry revealed only mild variations between the features of their profiles

6618 J. Phys. Chem. B, Vol. 112, No. 21, 2008 without and with G. Lower alkyl chain hydrophobicity has thus manifested a lower degree of interaction. Conclusion The interaction of ATABs with G at pH 9 and µ ) 0.005 proceeds through stagewise changes whose probing is methoddependent, and their rationalization with a unique model is a challenging task. The intermediate stages for the two higher homologues CTAB and OTAB can be several; initial GSIn gives B way to GSm , a necklace bead-type patterned aggregate. The complex prior to its saturated state undergoes self-aggregation to form coacervates that reach to a state of phase separation/ precipitation. This GSCm subsequently is disintegrated in the free micellar solution of ATAB. In excess ATAB solution, free monomer, the necklace bead complex, and free micelles exist together in solution controlled by several thermodynamic equilibria. Lower homologues with decreased alkyl chain length, namely, TTAB and DTAB, undergo weaker interaction with G. The interfacial properties of the G-ATAB system can be a good guide to the interaction process, which is influenced by the bulk processes as well. Temperature augments greater interaction, favoring a disaggregation and dissolution of the coacervates. The effective charge on G is a determinant factor for the G-ATAB (more precisely G-amphiphile) interaction. Such studies, as well as the investigation in the presence of salts, are needed for a better understanding of the G-amphiphile system in terms of topological and morphological standpoints. We are in the process of examining the variation of the pH of the medium on the interaction of G with CTAB and its headgroup analogues and the morphologies and structures of the G-CTAB complexes by SEM and AFM techniques. The results will be presented in a forthcoming paper. Acknowledgment. D. M. thanks the Council of Scientific and Industrial Research, Government of India, for financial support. S. P. M. thanks the Jadavpur University for an Emeritus Professorship and the Indian National Science Academy for an Honorary Scientist position. We thank Prof. Amitava Patra of IACS, Kolkata, for fluorescence measurements. Supporting Information Available: The phase and the interfacial adsorption behavior of G at various concentrations and temperatures are presented in Tables S I and II. Micellization of pure ATAB and related parameters are illustrated in Table S III. Also, relevant equations for deducing interfacial and bulk parameters are discussed in the footnotes. Interfacial and bulk properties for the G-ATAB interaction are presented in Table S IV. This information is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Ananthapadmanabhan, K. P. In Interactions of Surfactants with Polymers and Proteins; Goddard, E. D., Ananthapadmanabhan, K. P., Eds.; CRC Press, Inc.: London, U.K., 1993; Chapter 8. (2) Jonsson, B.; Lindman, B.; Holmberg, K.; Kronberg, B. Surfactants and Polymers in Aqueous Solutions; Wiley and Sons: New York, 1998. (3) (a) Reynolds, J. A.; Tanford, C. J. Biol. Chem. 1970, 245, 5161. (b) Tanford, C. J. Mol. Biol. 1972, 67, 59. (4) Decker, R. V.; Foster, J. F. Biochemistry 1966, 5, 1242. (5) Jones, M. N.; Manley, P. J. Chem. Soc., Faraday Trans. 1980, 76, 654. (6) (a) Leal, C.; Moniri, E.; Pegado, L.; Wennerstrom, H. J. Phys. Chem. B 2007, 111, 5999. (b) Taylor, D. J. F.; Thomas, R. K.; Penfold, J. AdV. Colloid Interface Sci. 2007, 132, 69. (c) Wang, X.; Wang, J.; Wang, Y.; Yan, H. Langmuir 2004, 20, 9014. (d) Asnacios, A.; Langevin, D.; Argillier, J.-F. Macromolecules 1996, 29, 7412. (e) Lim, P. F. C.; Chee, L. Y.; Chen, S. B. J. Phys. Chem. B 2003, 107, 6491. (f) Klebanau, A.; Kliabanova, N.; Ortega, F.; Monroy, F.; Rubio, R. G.; Starov, V. J. Phys.

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