Interaction of Gelatin with Room Temperature Ionic Liquids: A Detailed

J. Phys. Chem. B 2010, 114, 8441–8448

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Interaction of Gelatin with Room Temperature Ionic Liquids: A Detailed Physicochemical Study Tejwant Singh,† Shilpi Boral,‡ H. B. Bohidar,‡ and Arvind Kumar*,† Central Salt and Marine Chemicals Research Institute, Council of Scientific & Industrial Research (CSIR), G. B. Marg, BhaVnagar-364002, Gujarat, India and Polymer and Biophysics Laboratory, School of Physical Sciences, Jawaharlal Nehru UniVersity, New Delhi-110 067, India ReceiVed: March 17, 2010; ReVised Manuscript ReceiVed: May 19, 2010

Interaction of gelatin (G) with room temperature ionic liquids (ILs), 3-methyl-1-octylimidazolium chloride [C8mim][Cl] and 1-butyl-3-methylimidazolium octylsulfate [C4mim][C8OSO3], have been investigated through tensiometry, conductivity, steady-state fluorescence, turbidity, and dynamic light scattering (DLS). We have observed that the nature of interactions in G-[C8mim][Cl] system are remarkably different as compared to G-[C4mim][C8OSO3] system. At low concentrations, much below the critical micelle concentration (cmc) of IL, the IL monomers are adsorbed at the native G at the interface forming G-IL (monomer) complex, whereas both the monomers and lower IL aggregates are interacted with G in bulk leading to G-IL (aggregate) complex. The increased hydrophobic character of the G-IL complexes is evidenced from pyrene fluorescence. Turbidity measurements showed interestingly distinguished coacervation characteristics in the investigated systems. In case of G-[C4mim][C8OSO3] system, the coacervates dissolve in the free micellar solution whereas G-[C8mim][Cl] coacervates remain stable up to very high concentration. DLS provided useful information about the changes in size of gelatin and the nature of interactions between gelatin and ILs. Thermodynamic parameters of micellization with and without gelatin have been derived and compared. 1. Introduction Gelatin (G) is a biodegradable model polypeptide polyampholyte derived from denatured collagen lacking any secondary and tertiary structure. At neutral pH, about 7.5% of the gelatin is positively charged (lysine and arganine), 12% is negatively charged (glutamic and aspartic acid), and 6% of the chain is hydrophobic (leucine, isoleucine, methionine, and valine) in nature leaving about 58% of the chain comprising of glycine, proline, and hydroxyproline to be neutral. Gelatin has a wide range of commercial applications in food, cosmetics, drug encapsulation, pharmaceutics, or photography industries because of the properties such as emulsifier, thickener, and peptizer.1-3 In several of these applications, gelatin is used in conjunction with various surfactants to provide emulsification capacity or to control interfacial tension. Hence, the G-surfactant interactions are of immense importance in various industrial processes. G-surfactant interactions have been widely studied for many years employing both classical and modern techniques.4-29 Mitra et al.4 investigated the interaction of gelatin with a cationic amphiphile, cetyltrimethylammonium bromide, CTAB, using a variety of techniques and concluded that the interaction proceeds through stagewise changes whose probing is method dependent, and their rationalization with a unique model is a challenging task. Griffiths et al.7-10 have studied the interaction of gelatin with alkyl sulfate surfactants and demonstrated the effect of alkyl chain length on G-alkyl sulfate interactions. The formation of amphiphillic and neutral aggregates in the mixtures of G-SDS has been reported by Onesippe et al.14 Evidence of the adsorption * To whom correspondence should be addressed. E-mail: mailme_arvind@ yahoo.com; [email protected]. Tel: +91-278-2567039. Fax: +91-2782567562. † Central Salt and Marine Chemicals Research Institute (CSIR). ‡ School of Physical Sciences, Jawaharlal Nehru University.

of micellar aggregates on gelatin has also been reported by various researchers.17-25 Greener et al.26studied the binding of gelatin to a variety of surfactants ranging from ionic to nonionic using viscometric methods and concluded that the extent of interaction was closely related to surfactant type, G-surfactant composition, and ionic strength of the solution with lesser effects exerted by gelatin type and pH level. Dynamic light scattering (DLS) study conducted by Saxena et al.27 revealed the presence of electrostatic interactions between polar groups of gelatin and ionic surfactants whereas in the case of nonionic surfactants the interactions are mainly hydrophobic in nature. Light scattering studies also revealed the complex formation of gelatin with negatively charged polyanions like sodium polystyrenesulfonate (NaPSS) or sodium poly(2- acrylamido-2-methylpropanesulfonate) governed by polarization induced attraction.28 All these studies indicate that G-surfactant interactions are controlled mainly by surfactant type (anionic, cationic, or nonionic), composition of G-surfactant mixture, and ionic strength. Many of the room temperature ionic liquids (ILs) are emerging as novel surfactants due to amphiphilic nature of their cation or anion, and have been explored for their aggregation behavior by many researchers including our own group.30-47 The most intensively studied ILs are based on imidazolium cation [Cnmim]. With the possibility of fine-tuning the hydrophobicity of ILs by changing the alkyl chain length, polarizability of headgroup, and the nature of the anions one can generate specific self-assembled structures. Moreover, some of the ILs have shown the superior surface activity as compared to conventional cationic surfactants.32 Unique physicochemical properties and superior surface activity of ILs can be exploited for utilizing these compounds as a potential substitute for conventional surfactants in various applications. Therefore, herein we investigate the interactions of ILs, [C8mim][Cl] and

10.1021/jp102419f  2010 American Chemical Society Published on Web 06/08/2010

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[C4mim][C8OSO3] with a model protein gelatin. Till now, ILsprotein interactions have been rarely studied. These studies focus on the effect of ILs on the conformation, aggregation, or oligomerization, stabilization and dynamics of enzyme/protein.48-55 Recently, Geng et al.,55 have studied the interaction of a surfactant like IL, 1-tetradecyl-3-methylimidazolium bromide [C14mim][Br] with bovine serum albumin using surface tension measurements and fluorescence spectroscopy. However, by and large the detailed and systematic studies on protein-IL interactions are still lacking. As far as gelatin is concerned, no information is available on G-ILs interactions in the open literature. Therefore present studies can be of immense importance for utilizing these combinations in various biotechnological processes. In brief, we have carried out a detailed physicochemical investigation on the interaction of gelatin with surfactant like ILs, [C8mim][Cl] and [C4mim][C8OSO3] at a pH 7.0 (lower than the isoelectric point, pH 9.0) in the phosphate buffer medium. The interfacial behavior of systems has been examined using tensiometry. Other techniques, such as conductivity, fluorescence spectroscopy, turbidity, and DLS have been used to understand G-IL interactions in bulk. The probing of the interaction process was found to be method dependent wherein the different methods showed different features of interaction process due to phenomenological differences in interaction at interface and in bulk. Fluorescence spectroscopy using pyrene as a polarity probe provided useful information about the polarity of microdomains in the G-IL complexes. The coacervate formation process has been monitored by turbidimetric profiles. DLS was used to elucidate the change in hydrodynamic radii of gelatin as a consequence of G-IL interactions at various IL concentrations. The DLS results have been explained by necklace-bead model of polymer-surfactant interactions.56 2. Experimental Section A. Materials. 3-Methyl-1-octylimidazolium chloride [C8mim][Cl] and 1-butyl-3-methylimidazolium octylsulfate [C4mim][C8OSO3] with stated purities higher than 98% mass fraction were purchased from Solvent Innovation, Germany. ILs were dried and degassed under vacuum at 60 °C for 2-3 days to remove moisture. Karl Fischer analysis of the samples indicated that the water content was reduced to less than 0.02%. Gelatin type A (300 Bloom, IEP ) 9.0) was procured from Sigma. Pyrene (Aldrich, U.S.A.) was used for fluorescence measurements. The solutions of ILs with or without gelatin were prepared by weight using an analytical balance with a precision of (0.0001 g (Denver Instrument APX-200) in phosphate buffer. The buffer solution was prepared in degassed Millipore grade water using AR-grade dihydrogen sodim phosphate and disodium hydrogen phosphate purchased from Merck, India. B. Methods. All measurements were performed in phosphate buffer solution at pH 7.0. Gelatin solutions were prepared by soaking the gelatin powder in buffer for 1 h followed by heating up to 45 °C with mild stirring. The gelatin concentration for the present study was kept 0.2 wt %. i. Tensiometry. Surface tension measurements were carried out at 298.15 K using a DataPhysics DCAT II automated tensiometer employing the Wilhelmy plate method. IL was added into the gelatin solution by weight and stirred for 2-3 min for complete solubilization. Prior to measurements the resultant solutions were kept for at least 10 min for equilibration. The data was collected in duplicate and was found to be accurate within (0.1 mN m-1. The temperature of the measurement cell was controlled with a Julabo water thermostat within (0.1 K.

Singh et al. ii. Conductometry. Electrical conductivities were measured at 298.15 K by a digital conductivity meter (Systronics 308) using a cell of unit cell constant. The temperature of the measurement cell was controlled with a Julabo water thermostat within (0.1 K. Measurements were performed with an uncertainty of less than 0.5%. iii. Turbidimetry. The shoulder in the spectra of gelatin solutions at λ 275 nm was employed to produce the turbidity index (100 - %T at 275 nm) using an UV-visible spectrophotometer model (Cary 500, Varian). The spectral measurements were taken in percent transmittance mode (%T) in the wavelength range 200-500 nm using a quartz cuvette having path length of 1 cm. The ILs were progressively added by weight to the sample cell containing 2.5 mL of the gelatin solution. The solutions were allowed to equilibrate for approximately 5 min after stirring. Buffer solutions of ILs were used as reference. iW. Fluorimetry. Steady-state fluorescence measurements were performed using a Fluorolog (Horiba Jobin Yvon) spectrometer using a quartz cuvette of path length 1 cm. Pyrene was used as the polarity probe as the ratio (I1/I3) of its first vibronic peak (373 nm) to third vibronic peak (384 nm) is very sensitive to the polarity of the surroundings. The emission spectra of pyrene was recorded in the wavelength range 350-500 nm at an excitation wavelength of 334 nm using the excitation and emission slit widths of 1 nm. The concentration of pyrene used was 2 µM. The fluorescence spectra were corrected for the instrumental response. W. Dynamic Light Scattering. The DLS measurements were performed at 298.15 K on a light scattering apparatus described elsewhere.27 Appropriate amount of ILs were added by weight to the gelatin solution (2 mL) taken in a cylindrical quartz cuvette. The concentration range was varied from very dilute to above critical micelle concentration (cmc). Temperature of the measurements was controlled with an accuracy of (0.1 K. 3. Results and Discussions A. Micellization of Pure ILs in Buffer at pH 7.0. i. Tensiometry. The tensiometric profiles of both the studied ILs in the pH 7 buffer are shown in Figure 1a,b (open symbols). In case of [C8mim][Cl], the mild surface activity was observed initially followed by a sharp decrease in surface tension (γ) up to completion of monolayer formation at γcmc (37.3 mN m-1) whereas γ decrease linearly for [C4mim][C8OSO3] up to γcmc (30.5 mN m-1). The surface activity of [C4mim][C8OSO3] is found to be more than [C8mim][Cl] as evidenced from the lower γcmc for the former. Higher surface activity for [C4mim][C8OSO3] is due to surface active nature of both the constituent ions. The γ of studied ILs showed a pronounced minima near cmc, after that γ subsequently increases to attain a constant value. Similar type of behavior for aqueous solutions of ILs was also observed by other researchers.34,37,57 The relative surface excess of water near the imidazolium cation leading to some structural changes as a consequence of anions occupying the surface near cmc may be the reason for such behavior. The Gibbs’ surface excess (Γmax), area of exclusion per monomer (Amin) and the standard Gibbs free energy of interfacial adsorption (∆G°ad) (using Gibbs free energy of micellization ∆G°m deduced from conductivity measurements) were calculated following the standard procedures and equations (see Supporting Information for relevant equations and references). The thermodynamic parameters thus obtained are given in Table 1. Higher value of Γmax and lower magnitude of Amin for [C4mim][C8OSO3] as compared to [C8mim][Cl] shows the greater efficacy of [C4mim][C8OSO3] to populate the surface.

Interaction of Gelatin with RT Ionic Liquids

Figure 1. Surface tension (γ) as a function of IL concentration: (a) [C8mim][Cl] and (b) [C4mim][C8OSO3]. Various transitions discussed in text are marked with vertical lines.

A comparison of ∆G°ad and ∆G°mic shows that the interfacial adsorption of ILs is more spontaneous than the micellization process. ii. Conductometry, Fluorimetry, and DLS. The break point in the profiles of specific conductance versus IL concentration shown in Figure 2a,b corresponds to onset of micellization in bulk at cmc. Standard free energy of micellization ∆G°m of studied ILs was derived by the application of the mass action model using the equation58 o ∆Gm ) (2 - R)RT ln Xcmc

where R is the degree of ionization that is computed from the ratio of slopes of the post micellization and premicellization regions of the specific conductance versus concentration profiles of the ILs and Xcmc is the mole fraction of ILs at cmc. Experimental points were fitted to a first degree polynomial with a correlation coefficient greater than 0.998. The cmc, R, and ∆G°m values are given in Table 1. cmc values obtained from conductometry are comparable to those observed from surface tension measurements. [C4mim][C8OSO3] micellize at lower concentration as compared to [C8mim][Cl]. Also, the higher value of ∆G°m in [C4mim][C8OSO3] is indicative of more spontaneous micellization as compared to [C8mim][Cl]. Steady state fluorescence spectroscopy was used to determine the cmc and microenvironment of the micelle of ILs in the solutions. Relative intensity of vibronic bands I1/I3 of pyrene fluorescence as a function of IL concentration is plotted in Figure

J. Phys. Chem. B, Vol. 114, No. 25, 2010 8443 3a,b (open symbols). The ratio I1/I3 remains constant up to a certain concentration and then decreases rapidly then again attains almost a constant value with further increases in IL concentration. The cmc values derived from the midpoint of transition in I1/I3 are given in Table 1. The vibronic structure of the fluorescence spectrum of monomeric pyrene is known to be sensitive to the local polarity. The ratio I1/I3 of solubilized pyrene increases on going from nonpolar to polar solvents.59,60 I1/I3 values for [C8mim][Cl] and [C4mim][C8OSO3] were found to be 1.56 and 1.39, respectively, and suggest a moderate polarity of cybotactic region as sensed by pyrene in these micelles. DLS measurements above cmc showed a hydrodynamic radii of approximately 1.5 nm for the micelles formed of [C8mim][Cl] and [C4mim][C8OSO3]. B. G-ILs Interactions in Buffer at pH 7.0. i. Tensiometry. Tensiometric profiles of [C8mim][Cl] and [C4mim][C8OSO3] in buffer solutions of 0.2% gelatin are compared in Figure 1a,b (solid symbols). High surface active nature of gelatin resulted in a decrease in the surface tension of solution as compared to buffer. Different interactional behavior of ILs toward gelatin has been observed from tensiometric isotherms. Various stages of G-IL interactions have been observed from tensiometry. A steep decrease in surface tension of gelatin solution upon addition of [C8mim][Cl] has been observed in the very dilute IL solutions due to formation of G-[C8mim][Cl] (monomer) complex at interface. The association of [C8mim]+ to the negatively charged acidic residues of gelatin at this pH forms a surface active species thereby reducing the γ up to C1. Such complexation is due to electrostatically driven cooperativity for strongly interacting systems4,61,62 though the role of hydrophobic interactions between alkyl chain of IL and hydrophobic parts of gelatin cannot be ruled out. With further addition of IL, γ remains constant in a narrow range up to C2 and then starts increasing. At C2, also called as critical aggregation concentration (cac) the [C8mim]+ started interacting with the gelatin chain in bulk forming small assemblies as a consequence of initial monomeric adsorption leading to increase in local IL concentration in vicinity of polymer backbone.63 At cac, the interfacially active G-[C8mim][Cl] (monomer) complex starts transforming into G-[C8mim][Cl] (aggregate) complex with a decreased interfacial activity as indicated from increase in surface tension (Figure 1a). At this point, a decrease in polarity is evidenced from fluorescence spectroscopy (discussed later) showing the formation of hydrophobic domains. Similar type of behavior was also observed in case of G-CTAB system.4 The γ increases up to C3 that may be due to the collapse of an IL decorated gelatin backbone from interface to bulk. The coacervation phenomenon has been initiated at C3 as evidenced by the increase in the turbidity index of the solution at around this concentration (discussed later). A second plateau between C3 and Cm has been observed. In this region, the smaller aggregates continue to bind with the polymer sites in bulk, restricting the IL monomers to adsorb at the interface and maintaining nearly a constant γ. At Cm, the binding of small micelle to gelatin was complete and the incoming monomers accumulate at the interface reducing γ up to C4 where free micelle begins to form in the solution. Contrary to G-CTAB system,4 the inflection point CT in turbidimetry, which corresponds to the formation of coacervates with distinct turbidity, was not observed in the tensiometric profiles of G-[C8mim][Cl] system. Contrary to other surfactant-biopolymer systems,4,64 the cmc (C4) has not been found to be extended in G-[C8mim][Cl] system signifying the absence of collapse of IL saturated polymer. The C1, C2, C3, Cm, and C4 values are reported in Table 2. Thermodynamic

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TABLE 1: Critical Micelle Concentration (cmc) from Surface Tension (ST), Conductometry (cond), and Fluorescence (flr)a ILs in Buffer cmc/mmol kg

-1

ST

cond

flr

R

∆G°m

γcmc

Γmax × 106

Amin

∆G°ad

[C8mim][Cl] [C4mim][C8OSO3]

170.2 23.0

174.2 32.8

184.8 29.9

0.34 0.64

–20.9 –32.2

37.3 30.5

1.24 1.63

1.33 1.02

–24.6 –38.7

[C8mim][Cl] [C4mim][C8OSO3]

181.9 32.8

184.1 23.7

169.7 24.1

ILs in 0.2% Gelatin Solution 0.46 –19.0 38.2 0.74 –31.6 32.2

0.76 1.22

2.19 1.36

–26.3 –41.7

a The degree of counterion binding (R), standard free energy of micellization (∆G°m), surface tension at cmc (γcmc), Gibbs’ surface excess (Γmax), area of area of exclusion per monomer (Amin), and standard free energy of adsorption (∆G°ad) at 298.15 K. ∆G°m and ∆G°ad are expressed in kJ mol-1, γcmc, Γmax, and Amin are expressed in mN m-1, mol m-2, and nm2 molecule-1, respectively.

Figure 2. Specific conductance (κ) as a function of IL concentration: (a) [C8mim][Cl] (without gelatin); (b) [C4mim][C8OSO3] (without gelatin); (c) [C8mim][Cl] (with gelatin); (d) [C4mim][C8OSO3] (with gelatin). Various transitions discussed in text are marked. Dashed lines correspond to second derivative of κ versus IL concentration.

parameters, that is, γcmc, Γmax, Amin, and ∆G°ad (using ∆G°mfrom conductivity) calculated for G-IL systems are compared in Table 1. In the G-[C8mim][Cl] system, the Amin is higher as compared to gelatin free [C8mim][Cl] system indicating the decrease in efficacy of [C8mim][Cl] to polpulate the interface in presence of surface active gelatin. ∆G°ad was also found to be higher in the system containing gelatin. The tensiometric profiles of G-[C4mim][C8OSO3] system are quite different from that observed for G-[C8mim][Cl]. Interactions in the G-[C4mim][C8OSO3] system are complex as both the cation and anion of [C4mim][C8OSO3] being amphiphillic in nature are susceptible to interact with gelatin. In this system, γ remains constant initially up to the critical aggregation concentration, (C1), followed by a decrease to reach a minima at C2. After C2 the γ increases to reach maxima (C3) and then decreases steeply to attain a minima at C4 where free micelle begins to form. The initial constant γ signifies the interfacially ineffective interactions between adsorbed gelatin and IL ions.

Thereafter, the decrease in γ indicates the onset of interfacial population corresponding to the monomeric IL ions adsorption on the oppositely charged sites of the gelatin forming an IL-G (monomer complex) at interface. The process continues until C2 where the interface is saturated by IL-G (monomer complex). Similar behavior has also been reported for the monomeric interaction prior to aggregate formation in NaCMC-CTAB system.65 [C8OSO3]- and [C4mim]+ both possessing amphiphilic character are capable of interacting with positively and negatively charged moieties on the gelatin backbone. This becomes more evident when we compare the ratio of cmc to cac in G-[C4mim][C8OSO3] system with the conventional G-surfactant systems having the same alkyl chain length such as G-SOS.66 The higher ratio of cmc to cac for G-[C4mim][C8OSO3] (25.0) as compared G-SDS (1.9) indicates the greater efficacy of [C4mim][C8OSO3] to interact with gelatin. γ starts increasing from C2 to attain maxima at C3, which is due to partial collapse of IL decorated gelatin chain from interface to bulk. After C3,

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Figure 3. I1/I3 as a function of IL concentration: (a) [C8mim][Cl] and (b) [C4mim][C8OSO3]. Various transitions are marked with vertical lines.

TABLE 2: The Various Transition Concentrations (mmol kg-1) Observed from Various Techniques in G-ILs Systems at 298.15 K ST

cond

turbidity

flr

G-[C8mim][Cl] C1 C2, cac C3 (Ct) C4 (cm)

6.1 7.9 17.6 181.9 (66.6)

6.4 9.2 21.9 184.1

23.5 (156.8)

24.4 169.7 (57.7)

G-[C4mim][C8OSO3] C1, cac C2 C3 C4

1.2 2.3 3.4 32.8

2.0 3.4 23.7

36.5

3.8 24.1

the IL ions progressively occupy the free interface thereby reducing the γ up to C4. The γ decrease in a nonlinear fashion due to interplay between hydrophobic interaction and adsorption of small IL aggregates associated with the gelatin backbone. Such behavior is also observed in conventional polymer surfactant systems.65,67 The cmc for [C4mim][C8OSO3] in the presence of gelatin has been found to be extended indicating the collapse of IL saturated polymer. The C1, C2, C3, and C4 values are reported in Table 2. Thermodynamic parameters γcmc, Γmax, Amin, and ∆G°ads(using ∆G°m) have been calculated and tabulated in Table 1. Similar to G-[C8mim][Cl] system, an increase in Amin has also been observed in G-[C4mim][C8OSO3] system when compared to the gelatin free system. The relative increase in magnitude of Amin for G-[C4mim][C8OSO3] system is much less (0.34 nm2/molecule) as compared to that for

G-[C8mim][Cl] system (0.86 nm2/molecule). This indicates that [C4mim][C8OSO3] is more effective in populating the interface as compared to [C8mim][Cl] in the presence of gelatin, which is due to simultaneous adsorption of [C8OSO3]- and [C4mim]+ at the interface. The increase of ∆G°ads in the presence of gelatin is also higher for [C4mim][C8OSO3] (3.0 kJ mol-1) as compared to [C8mim][Cl](1.7 kJ mol-1). ii. Conductometry. Figure 2c,d shows the conductometric profiles of [C8mim][Cl] and [C4mim][C8OSO3] in buffer solutions of 0.2% gelatin, respectively. In the case of G-[C8mim][Cl] system three distinct transitions have been observed. These transitions fairly agreed with the concentrations: C2(cac), C3, and C4 (cmc) observed in the tensiometric profiles. Initially a small decrease in the specific conductance up to C2 has been observed indicating the adsorption of monomeric [C8mim]+ onto the gelatin in this region followed by steep increase up to C3 where the coacervation starts. In the conductometric profile, the C3 is a faint transition showing that the binding process in this region does not affect the conductance. The formation of free micelles in the solution starts at C4 where a prominent transition has been observed. The condensation of [Cl]- to the stern layer of the micelle leads to lowering in specific conductivity. The break points C1 and Cm observed in tensiometry were not observed in conductometry. Similar to conventional surfactantpolymer4,67 systems, the degree of counterion binding was found to be decreased in G-[C8mim][Cl] system (0.34) when compared to gelatin free system (0.46). The ∆G°mwas found to be less in the presence of gelatin as compared to gelatin-free system indicating that coacervation is a favorable phenomenon over the free micelle formation. In the conductometric profile of G-[C4mim][C8OSO3], two break points have been observed (Figure 2d). The first break point at very low concentration is consistent with the C3 observed from tensiometry. Initially, the conductance increases with a lower slope up to C3 and then follows a higher slope until C4. The low slope before C3 indicates the dominance of adsorption of IL ions onto gelatin both at the interface and bulk. Beyond C3 further formation and binding of small induced aggregates to gelatin backbone is limited and the IL prevails only as ions producing a linear conductance with higher slope up to C4. The break point at C4 corresponds to free micelle formation indicated by a drastic decrease of slope in specific conductance. Similar to the G-[C8mim][Cl] system, the degree of counterion binding was found to be decreased in G-[C4mim][C8OSO3] system (0.64) when compared to gelatin free system (0.74). iii. Fluorimetry. G-IL interactions were revealed by determining the polarity changes in the IL-gelatin solutions using pyrene fluorescence. Relative intensity of vibronic bands I1/I3 of pyrene fluorescence in G-[C8mim][Cl] and G-[C4mim][C8OSO3] solutions as a function of IL concentration is plotted in Figure 3a,b, respectively (solid symbols). As can be seen in Figure 3, in the presence of gelatin the addition of ILs ([C8mim][Cl] and [C4mim][C8OSO3]) decrease I1/I3 right from the beginning up to C1. The decrease of I1/I3 is due to formation of G-IL monomer complex with decreased hydrophilic character. In the case of the G-[C8mim][Cl] system, further addition of IL did not affect the I1/I3 up to C3, after which it increased until Cm. In this region, the polarity behavior is slightly different from interfacial behavior where the γ remained constant from C1 to C2 and increased continuously up to C3. Coacervates at Cm with a complete necklace bead type structure are most hydrophilic in nature. I1/I3 decreased continuously after Cm going through C4 before attaining a plateau at higher concentration. In the

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Figure 4. Turbidity index as a function of IL concentration: (a) [C8mim][Cl] and (b) [C4mim][C8OSO3]. Various transitions are marked with vertical lines.

system G-[C4mim][C8OSO3], I1/I3 starts increasing immediately after C1 reaching a maxima at C3 and then decreases slightly before a steep decrease. The I1/I3 maxima at C3 is indicative of formation of highly polar structures (coacervates) induced by adsorption of IL ions and lower IL aggregates onto gelatin backbone. Free micellar concentration C4 observed for both the systems in fluorometry is compared with that obtained from other techniques in Table 2. iW. Turbidimetry. Turbidimetric profiles of [C8mim][Cl] and [C4mim][C8OSO3] in buffer solutions of 0.2% gelatin are shown in Figure 4a,b, respectively. For the G-[C8mim][Cl] system, a nearly sigmoidal curve has been observed. At lower IL concentrations, the turbidity remains almost constant and then increases sharply to attain a plateau. An inflection point has

been observed at a concentration of 23.5 mmol kg-1, which is very close to C3 as observed from tensiometry and conductometry. This inflection point indicates the onset of coacervation. After C3, the growth of coacervates leads a steep increase in turbidity which maximizes at CT. The increase in size of coacervates after C3 is also evidenced from DLS data discussed latter. After CT, the turbidity decreased very slightly. Such behavior is not frequently observed and turbidity is reported to be declined as a consequence of disintegration and solubilization of formed coacervates by the free micelle.4,64,65,68-70 No significant decrease in turbidity post CT shows that the coacervates formed of G-[C8mim][Cl] are quite stable even in the presence of free IL micelles. Similar findings have also been reported for the inulin-OTAB system.71 The G-[C4mim][C8OSO3] system shows a different turbidimetric behavior as compared to G-[C8mim][Cl]. The turbidity decreases initially up to 18.5 mmol kg-1 after which it increases up to 36.0 mmol kg-1 (≈C4) and then again decreases. The initial decrease in turbidity with increase in IL concentration is indicative of contraction of the gelatin chain to form more compact structure. The increase in turbidity from 18.5 to 36.0 mmol kg-1 is due to expansion of gelatin chain as a consequence of growth of coacervates. The turbidity decrease after C4 is perhaps due to solubilization of G-[C4mim][C8OSO3] coacervates in the free micelles of IL. W. Dynamic Light Scattering. Variation in hydrodynamic radii (Rh) of gelatin chain as a consequence of G-IL interactions and free IL micelles in G-IL solutions as a function of IL concentration has been investigated through DLS measurements. The obtained autocorrelation function has been processed using a method described by Saxena et al.27 The data has been analyzed in terms of necklace-bead model of polymer-surfactant interactions.56 Below cmc, the free micelles of IL does not exist, hence the obtained correlation function was fitted to a single exponential fitting. After cmc, the single exponential fitting was not found to be adequate and we resorted to double-exponential fitting as done elsewhere.27 The obtained hydrodynamic radii (Rh) and the polydispersity (Pi) are given in Table 3. Variation of Rh for G-[C8mim][Cl] system as a function of IL concentration is shown in Figure 5a. A small increase in Rh has been observed up to C3. This increase in size is due to expansion of gelatin chain upon interaction with IL ions forming G-[C8mim][Cl] (monomer) or G-[C8mim][Cl] (aggregate) complex. The interactions between gelatin and [C8mim][Cl] are governed both by electrostatic and hydrophobic forces. The electrostatic interactions between gelatin and [C8mim]+ are comparatively weaker due to delocalization of the positive charge onto large amphiphillic imidazolium ring, therefore

TABLE 3: Hydrodynamic Radii of Gelatin (Rh) and Free Micelle Rh(mic) along with Polydispersity Index (P) As a Function of IL Concentration in the G-IL Systems conc/mmol kg-1 0.0 11.2 18.2 25.3 52.3 76.0 134.2 175.4 273.8 352.2 444.8 583.4

Rh/nm G-[C8mim][Cl] 21.3 ( 0.1 23.2 ( 0.1 22.7 ( 0.1 24.0 ( 0.1 32.0 ( 0.2 33.4 ( 0.2 43.4 ( 0.2 54.8 ( 0.5 58.0 ( 0.5 61.2 ( 0.5 62.5 ( 1.0 64.6 ( 1.0

Rh(mic)/nm

P

conc/mmol kg-1

1.4 ( 0.05 1.4 ( 0.05 1.5 ( 0.05 1.5 ( 0.7 1.6 ( 0.7

0.21 0.23 0.22 0.25 0.27 0.26 0.28 0.32 0.35 0.25 0.34 0.31

0.0 7.5 16.5 19.5 23.7 36.2 59.0 82.5 114.2 146.0 190.5

Rh/nm

Rh(mic)/nm

G-[C4mim][C8OSO3] 20.1 ( 0.1 17.4 ( 0.1 14.4 ( 0.1 18.2 ( 0.1 19.8 ( 0.1 22.1 ( 0.2 1.4 ( 0.05 22.8 ( 0.2 1.4 ( 0.05 23.2 ( 0.2 1.4 ( 0.05 23.0 ( 0.2 1.4 ( 0.05 22.8 ( 0.5 1.4 ( 0.05 22.5 ( 0.5 1.4 ( 0.05

P 0.24 0.28 0.27 0.32 0.18 0.26 0.24 0.28 0.36 0.28 0.30

Interaction of Gelatin with RT Ionic Liquids

J. Phys. Chem. B, Vol. 114, No. 25, 2010 8447 size of G-IL complex. After a concentration of 18.0 mmol kg1-, Rh increases up to 36.0 mmol kg1- (C4) due to growth of coacervates. At concentrations above C4 where free micelles exist, the Rh slightly decreases. 4. Conclusion We have studied different stages of the interaction process of gelatin with room temperature ionic liquids (ILs): 3-methyl1-octylimidazolium chloride [C8mim][Cl] and 1-butyl-3-methylimidazolium octylsulfate [C4mim][C8OSO3] using various techniques. Initially, the IL monomers interact with gelatin at interface to form G-IL (monomer) complex after which a poor interfacially active complex comprised of G-IL (aggregates) has been observed. Fluorescence spectroscopy indicates the presence of hydrophobic microdomains as a consequence of G-IL complex formation around critical aggregation concentration (cac). G-IL complexation leads to coacervation that is more pronounced in the case of G-[C8mim][Cl] as compared to G-[C4mim][C8OSO3] system. The hydrodynamic radii of gelatin increased rapidly with addition of [C8mim][Cl] until C4 (cmc), thereafter only a little increase in Rh was observed whereas the addition of [C4mim][C8OSO3] resulted in contraction of gelatin initially and then lead to an increase of size until C4(cmc). In both the systems, after C4 free micelles of nearly a constant size (∼1.5 nm) were found existing along with the G-IL complexes. Acknowledgment. The financial support by the Department of Science and Technology (DST), Government of India (Project No. SR/S1/PC-55/2008) is highly acknowledged. T.S. is thankful to CSIR, Government of India for award of SRF.

Figure 5. Hydrodynamic radius (Rh) as a function of IL concentration: (a) [C8mim][Cl] and (b) [C4mim][C8OSO3]. Various transitions are marked with vertical lines. Hollow symbols represents free IL micelles in G-IL solutions. +

adsorption of [C8mim] onto gelatin chain is not effective in neutralizing the charge. Consequently, no contraction of gelatin chain is observed upon G-[C8mim][Cl] (monomer) complex formation. Increase of G-[C8mim][Cl] (monomer) complex size could be due to electrostatic repulsion between positive sites of gelatin chain and cationic head groups of [C8mim][Cl]. After C3, the Rh of the G-[C8mim][Cl] complex increased at a faster rate up to C4, thereafter only a little increase in Rh was observed with increase in IL concentration. The rapid increase in Rh after C3 is due to formation and growth of G-[C8mim][Cl] (aggregate) complex (coacervates). Beyond C4, the growth of G-[C8mim][Cl] (aggregate) complex is limited indicating the stabilization of these complexes even in the presence of free micelles, which is also evidenced from turbidimetry. After C4, the free micelles and G-[C8mim][Cl] (aggregate) complex coexist. The Rh of the free micelle was approximately 1.4 nm at C4, which remained nearly constant at still higher IL concentrations. In the system G-[C4mim][C8OSO3], the Rh decreases initially, reaches a minima, and then increases steeply to attain a plateau. Variation of Rh as a function of IL concentration is shown in Figure 5b. Rh decreases about 30% up to 18.0 mmol kg1indicating the contraction of the gelatin chain to form a more compact structure. The contraction of gelatin chain is due to the reduced electrostatic repulsion between charged sites of gelatin as a consequence of G-IL interactions. The contraction leads to chain coiling bringing the hydrophobic tails of the IL closer and inducing micellization, which further decrease the

Supporting Information Available: Additional equations and references. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Whitesides, T. H.; Miller, D. D. Langmuir 1994, 10, 2899. (2) Rose, P. I. G. In The Theory of the Photographic Process, 4th ed.; James, T. H., Ed.; Macmillan: New York, 1977; p 51. (3) Howe, A. M. Curr. Opin. Colloid Interface Sci. 2000, 5, 288. (4) Mitra, D.; Bhattacharya, S. C.; Moulik, S. P. J. Phys. Chem. B 2008, 112, 6609. (5) Maulik, S.; Dutta, P.; Chattoraj, D. K.; Moulik, S. P. Colloid Surf. B 1998, 11, 1. (6) Chatterjee, A.; Moulik, S. P.; Majhi, P. R.; Sanyal, S. K. Biophys. Chem. 2002, 98, 313. (7) Griffiths, P. C.; Stilbs, P.; Howe, A. M.; Cosgrove, T. Langmuir 1996, 12, 2884. (8) Griffiths, P. C.; Fallis, I. A.; Teerapornchaisit, P.; Grillo, I. Langmuir 2001, 17, 2594. (9) Griffiths, P. C.; Cheung, A. Y. F.; Jenkins, R. L.; Howe, A. M.; Pitt, A. R.; Heenan, R. K.; King, S. M. Langmuir 2004, 20, 1161. (10) Griffiths, P. C.; Roe, J. A.; Bales, B. L.; Pitt, A. R.; Howe, A. M. Langmuir 2000, 16, 8248. (11) Wu, D.; Xu, G.; Sun, Y.; Zhang, H.; Mao, H.; Feng, Y. Biomacromolecules 2007, 8, 708. (12) Vinetsky, Y.; Magdassi, S. Colloid Polym. Sci. 1998, 276, 395. (13) Sovilj, V. Colloid Polym. Sci. 1998, 276, 328. (14) Onesippe, C.; Lagerge, S. Colloid Surf. A 2009, 337, 61. (15) Wu, D.; Xu, G.; Feng, Y.; Li, Y. Int. J. Biol. Macromol. 2007, 40, 345. (16) Cooke, D. J.; Dong, C. C.; Thomas, R. K.; Howe, A. M.; Simister, E. A.; Penfold, J. Langmuir 2000, 16, 6546. (17) Cosgrove, T.; White, S. J.; Zarbakhsh, A.; Heenan, R. K.; Howe, A. M. Langmuir 1995, 11, 744. (18) Cosgrove, T.; Hone, J. H. E.; Howe, A. M.; Heenan, R. K. Langmuir 1998, 14, 5376. (19) Kragh, A. M. Trans. Faraday Soc. 1964, 60, 225. (20) Knox, W. J.; Parshall, T. O. J. Colloid Interface Sci. 1970, 33, 16. (21) Wustneck, R.; Hernel, H.; Kretzschmar, G. J. Colloid Interface Sci. 1983, 93, 419.

8448

J. Phys. Chem. B, Vol. 114, No. 25, 2010

(22) Buron, C.; Filiatre, C.; Membrey, F.; Foissy, A.; Argillier, J. F. Colloid Polym. Sci. 2004, 282, 446. (23) Wustneck, R.; Warnheim, T. Colloid Polym. Sci. 1988, 266, 926. (24) Isemura, T.; Tokiwa, F.; Ikeda, S. Bull. Chem. Soc. Jpn. 1962, 35, 240. (25) Arora, J. P. S.; Soam, D.; Singh, S. P.; Kumar, R. Tenside Deterg. 1984, 21, 2. (26) Greener, J.; Contestable, B. A.; Bale, M. D. Macromolecules 1972, 20, 2490. (27) Saxena, A.; Antony, T.; Bohidar, H. B. J. Phys. Chem. B 1998, 102, 5063. (28) Bowman, W. A.; Rubinstein, M.; Tan, J. S. Macromolecules 1997, 30, 3262. (29) Rao, A.; Kim, Y.; Kausch, C. M.; Thomas, R. R. Langmuir 2006, 22, 7964. (30) Singh, T.; Kumar, A. J. Phys. Chem. B. 2008, 112, 4079. (31) Singh, T.; Kumar, A. Colloid Surf. A 2008, 318, 263. (32) Dong, B.; Li, N.; Zheng, L.; Yu, L.; Inoue, T. Langmuir 2007, 23, 4178. (33) Dong, B.; Zhao, X. Y.; Zheng, L. Q.; Zhang, J.; Li, N.; Inoue, T. Colloid Surf. A 2008, 317, 666. (34) Goodchild, I.; Collier, L.; Millar, S. L.; Prokesˇ, I.; Lord, J. C. D.; Butts, C. P.; Bowers, J.; Webster, J. R. P.; Heenan, R. K. J. Colloid Interface Sci. 2007, 307, 455. (35) Wang, J.; Wang, H.; Zhang, S.; Zhang, H.; Zhao, Y. J. Phys. Chem. B 2007, 111, 6181. (36) Zhao, Y.; Gao, S. J.; Wang, J. J.; Tang, J. M. J. Phys. Chem. B 2008, 112, 2031. (37) Bowers, J.; Butts, P.; Martin, J.; Vergara-Gutierrez, C.; Heenan, K. Langmuir 2004, 20, 2191. (38) Inoue, T.; Ebina, H.; Dong, B.; Zheng, L. Q. J. Colloid Interface Sci. 2007, 314, 236. (39) Wu, B.; Zhang, L.; Zhang, Y. M.; Wang, H. P. Chem.sEur. J. [Online early access]. DOI: 10.1002/chem.200901098. (40) Dorbritz, S.; Ruth, W.; Kragl, U. AdV. Synth. Catal. 2005, 347, 1273. (41) Miskolczy, Z.; Sebok-Nagy, K.; Biczok, L.; Goektuerk, S. Chem. Phys. Lett. 2004, 400, 296. (42) Seth, D.; Sarkar, S.; Sarkar, N. Langmuir 2008, 24, 7085. (43) Dong, B.; Gao, Y.; Su, Y.; Zheng, L.; Xu, J.; Inoue, T. J. Phys. Chem. B 2010, 114, 340. (44) Dong, B.; Zhang, J.; Zheng, L. Q.; Wang, S. Q.; Li, X. W.; Inoue, T. J. Colloid Interface Sci. 2008, 319, 338. (45) Wang, H. Y.; Wang, J. J.; Zhang, S. B.; Xuan, X. P. J. Phys. Chem. B 2008, 112, 16682. (46) Blesic, M.; Lopes, A.; Melo, E.; Petroversuski, Z.; Plechkova, N. V.; Canongia Lopes, J. N.; Seddon, K. R.; Rebelo, L. P. N. J. Phys. Chem. B 2008, 112, 8645.

Singh et al. (47) Blesic, M.; Marques, M. H.; Plechkova, N. V.; Seddon, K. R.; Rebelo, L. P. N.; Lopes, A. Green Chem. 2007, 9, 481. (48) Laszlo, J. A.; Compton, D. L. Biotechnol. Bioeng. 2001, 75, 181. (49) Eckstein, M.; Sesing, M.; Kragl, U.; Adlercreutz, P. Biotechnol. Lett. 2002, 24, 867. (50) Lou, W. Y.; Zong, M. H.; Smith, T. J.; Wu, H.; Wang, J. F. Green Chem. 2006, 8, 509. (51) Zhao, H. J. Mol. Catal. B 2005, 37, 16. (52) Zhao, H.; Campbell, S. M.; Jackson, L.; Song, Z.; Olubajo, O. Tetrahedron: Asymmetry 2006, 17, 377. (53) Constantinescu, D.; Weinga¨rtner, H.; Herrmann, C. Angew. Chem., Int. Ed. 2007, 46, 8887. (54) Baker, G. A.; Heller, W. T. Chem. Eng. J. 2009, 147, 6. (55) Geng, F.; Zheng, L.; Yu, Li.; Li, G.; Tung, C. Process Biochem. 2010, 45, 306. (56) Ananthapadmanabhan, K. P. In Interactions of Surfactants with Polymers and Proteins; Goddard, E. D., Ananthapadmanabhan, K. P., Eds.; CRC Press: Boca Raton, 1993; p 319. (57) Sung, J.; Jeon, Y.; Kim, D.; Iwahashi, T.; Iimori, T.; Seki, K.; Ouchi, Y. Chem. Phys. Lett. 2005, 406, 495. (58) Rosen, M. J. Surfactant and Interfacial Phenomenon, 2nd ed.; John Wiley and Sons: New York, 1988. (59) Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. Soc. 1977, 99, 2039. (60) Nakajima, A. Bull. Chem. Soc. Jpn. 1971, 44, 3272. (61) Asnacios, A.; Langevin, D.; Argillier, J. F. Macromolecules 1996, 29, 7412. (62) Staples, E.; Tucker, I.; Penfold, J.; Warren, N.; Thomas, R. K.; Taylor, D. J. F. Langmuir 2002, 18, 5147. (63) Wang, C.; Tam, K. C. Langmuir 2002, 18, 6484. (64) Chakraborty, T.; Chakraborty, I.; Moulik, S. P.; Ghosh, S. J. Phys. Chem. B 2007, 111, 2736. (65) Chakraborty, T.; Chakraborty, I.; Ghosh, S. Langmuir 2006, 22, 9905. (66) Griffiths, P. C.; Stilbs, P.; Howe, A. M.; Whitesides, T. H. Langmuir 1996, 12, 5302. (67) Cabane, B.; Duplessix, R. J. J. Phys. (Paris) 1982, 43, 1529. (68) Dan, A.; Ghosh, S.; Moulik, S. P. Carbohydr. Polym. 2010, 80, 44. (69) Lundin, M.; Macakova, L.; Dedinaite, A.; Claesson, P. Langmuir 2008, 24, 3814. (70) Bu, H.; Kjøniksen, A.-L.; Elgsaeter, A.; Nystro¨m, B. Colloid Surf. A 2006, 278, 166. (71) Dan, A.; Ghosh, S.; Moulik, S. P. J. Phys. Chem. B 2009, 113, 8505.

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