Journal of Colloid and Interface Science 395 (2013) 135–144

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Journal of Colloid and Interface Science

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Amphiphilic behavior of two phosphonium based ionic liquids

Indrajyoti Mukherjee, Suvasree Mukherjee, Bappaditya Naskar, Soumen Ghosh, Satya P. Moulik ⇑Centre for Surface Science, Department of Chemistry, Jadavpur University, Kolkata 700 032, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 8 October 2012Accepted 28 November 2012Available online 19 December 2012

Keywords:Ionic liquidsViscositySurface activityMicellesMicroemulsionsPhase behaviorAnti-cancer activity

0021-9797/$ - see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.jcis.2012.11.064

⇑ Corresponding author. Fax: +91 33 2414 6266.E-mail address: spmcss@yahoo.com (S.P. Moulik).

Solution and surface chemical behavior of two phosphonium based ionic liquids triisobutyl (methyl)phosphonium tosylate (IL-1) and trihexyl (tetradecyl) phosphonium bis 2,4,4-(trimethylpentyl)phosphi-nate (IL-2) have been studied. The polar IL-1 is surface active and water soluble, whereas the weakly polarIL-2 is more surface active with very low aqueous solubility. IL-1 does not form micelles but affects themicellization properties of ionic, nonionic, and zwitterionic surfactants more strongly than conventionalelectrolytes. IL-2 itself forms micelles and mixed micelles with Triton X-100 (TX-100) in aqueous solu-tion. It also forms Langmuir monolayers of liquid expanded type, at the air/water interface. IL-1 canreplace water in forming microemulsions with the oil isopropylmyristate (IPM), stabilized by IL-2 (sur-factant) + isopropanol (IP as a co-surfactant) like the IL-1/IPM/(IL-2+IP) system. It produces a large mon-ophasic zone in the pseudoternary phase diagram. The thermodynamics of formation of themicroemulsions of IL-1 in oil (IPM) have been examined. The dimensions and the polydispersity of thedispersed nano-droplets in the microemulsions have been determined by DLS. The thermal stability ofthe microemulsion forming systems has also been studied. ILs studied against Sarcoma-180 cell lineshave evidenced proficient anti-cancer activity of IL-1 and moderate activity of IL-2.

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1. Introduction

Room temperature ionic liquids (RTILs) are important for theirconvenient physicochemical properties like low vapor pressure,low melting point (<100 �C) [1], non-flammability [2], high con-ductivity [3], good thermal and chemical stability [4,5], tunablesolubility, etc. ILs comprise bulky asymmetric organic cations (thatprevent crystal packing) and either inorganic or organic anions(with delocalized electron clouds that reduce inter-ionic interac-tion). ILs have potential in all fields of chemical studies, in variousindustrial applications [6,7] viz., fuel, photochemical, andrechargeable lithium cells [8], various electrochemical devices,etc. [9]. Investigations into biodegradation and antimicrobial activ-ities of ILs are getting importance [10]. Reports on anti-canceractivity of phosphonium and ammonium based ionic liquids arefound in literature [11,12]. ILs are not as green as was initially con-sidered. The environmental, health, and safety impact of ILs hasbeen a growing concern; evaluation of their toxicity levels is beingconsidered important [13]. ILs with long alkyl side chains are foundto be cytotoxic. Hexafluoro phosphate and tetrafluoro borate basedILs can undergo thermal and chemical decomposition and in acidscan undergo hydrolysis forming HF at low temperature [14]. Differ-ent types of adverse effects may be produced by many other ILs.Interestingly, choline based ILs have prospects in polymerization

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to eliminate toxic initiators and solvents, in the preparation ofmesoporous biocompatible silica aerogel, in biodegradation of dyesand as nutrients for growing bacteria, etc. [15–18].

Amphiphile behavior of ILs has become an attractive area of re-search. ILs comprising alkyltriphenylphosphonium cation withlong alkyl chains as surfactants for cationic exchange of layered sil-icates have been reported [19]. Phosphonium based ILs can appre-ciably lower surface tension (c) of water to 30–35 mN m�1 at�298 K showing potential amphiphilicity to form micelles [20–22]. ILs are also known to have significant influence on self-associ-ation of ionic, nonionic and zwitterionic amphiphiles [23–25]. Theycan also form stable microemulsions [26,27]. Rojas et al. [28] stud-ied microemulsions comprising IL, 1-ethyl-3-methylimidazoliumn-hexylsulfate–water (1:1)/toluene–pentanol (1:1)/SDS and CTAB.1-ethyl-3-methylimidazolium tetrafluoroborate (polar phase)/IPM–(oil)/(Tween-80 + Span-20) employed microemulsion hasbeen reported to be a proficient vehicle for encapsulation of differ-ent kind of drugs [29]. Zwitterionic, nonionic, and anionic surfac-tants with hydrophobic IL (oil) have been used to form oil/watermicroemulsions [30,31]. Replacement of water by IL can increasethermal stability range of microemulsions [32]. Properties ofnon-aqueous micelles and microemulsions with ILs have beencomprehensively compared with their aqueous counterparts [33–35].

The two phosphonium cation based ILs herein studied (shownin Scheme 1) have higher thermal and electrochemical stabilitythan their ammonium analogs. Their properties have been only

Scheme 1. (a and b) Molecular structures of IL-1 and IL-2.

136 I. Mukherjee et al. / Journal of Colloid and Interface Science 395 (2013) 135–144

limitedly studied [36–38]. IL-1 is a good carrier of CO2 in its aque-ous solution than water [39]; IL-2 is a prospective solvent for sep-arating aromatic hydrocarbons from aliphatics [40]. Phosphoniumbased ILs are effective carriers of Boranes [41]. IL-2 type com-pounds have uses in nanoparticle synthesis, adamantane solubili-zation, etc. [42,43].

Our work plan comprised investigations into the interfacial andbulk properties of IL-1 and IL-2 separately as well as in mixtureswith conventional amphiphiles. We have examined surface activityof both the ILs, especially Langmuir monolayer properties of IL-2.The former has good surface activity but does not self assemble,whereas the latter is weakly polar and possesses significant surfaceactivity and proficiently self aggregate in aqueous solution. Themixed micelle formation of IL-2 with TX-100 and the influence ofIL-1 on the self-aggregation of conventional surfactants (TX-100,DTAB, SDS, and CHAPS) have been investigated. Phase behaviorsof the systems, water/IPM/IL-2+IP designated as (A), and IL-1/IPM/IL-2+IP or Bu designated as (B) or (B1) and dimensions ofthe dispersed droplets were determined. The information foundfrom tensiometry, conductometry, microcalorimetry, and dynamiclight scattering methods is comprehensively discussed in what fol-lows. The proficiency of the two ILs as anti-cancer agents has beenexamined; in a previous publication [44], antibacterial effects of IL-1 and IL-2 were reported.

2. Experimental section

2.1. Materials

The room temperature ionic liquids used were triisobu-tyl(methyl)phosphonium tosylate (IL-1), MW = 388.54; CAS No.[344774-05-6] and trihexyl(tetradecyl)phosphonium bis 2,4,4-(trimethylpentyl)phosphinate (IL-2), MW = 773.27; CAS No.[465527-58-6] from Fluka-Switzerland, and were used as received.They were >95.0% pure (31P NMR) [4] and herein characterized by1H NMR (300 MHz in CDCl3); results are presented in Text S-1A insupporting material or SM.

Water contents of IL-1, IL-2 found by Karl–Fischer titrationmethod (Model-331, Systronics-India) were 0.0143 and 0.0221mass fractions, respectively. The values were considered in theirsolution preparation. The greater water content of IL-2 than IL-1was due to hydrophobic hydration through its many methylenegroups compared to IL-1 (their respective viscosities are 0.3 and0.089 Pa s at zero shear and 298 K [44]).

The studied IL-1 and IL-2 are non-Newtonian fluids and evi-denced both shear thinning and thickening behaviors. Their flowbehaviors and related energetic parameters along with TX-100have been reported by us earlier [44].

The surfactants used were sodium dodecylsulfate (SDS) anddodecyltrimethylammonium bromide (DTAB) of SRL (India), 3-

[(3-cholamidopropyl) dimethylammonio] propanesulphonate(CHAPS) of Dojindo Laboratories (Japan), and p-tert-octylphenoxy-polyoxyethylene (9.5) ether (TX-100) of BDH (England). Their CMCvalues (given in Table 1) were in good agreement with the reportedvalues. Isopropylmyristate (IPM) used was of Fluka (Switzerland);isopropanol (IP) and butanol (Bu) were of SRL (India) and Merck(India), respectively. Trypan Blue for microscopy was purchasedfrom LOBA-CHEMIE (Mumbai, India).

The water used in the study was doubly distilled conductivitywater (specific conductance j = 2–4 lS cm�1 at 303 K).

2.2. Methods

Instrumental methods like 1H NMR, tensiometry (ST), conduc-tometry (cond.), microcalorimetry (lcal), dynamic light scattering(DLS) were employed in this study. Brief descriptions of the meth-ods are presented below.

2.2.1. 1H NMR1H NMR Spectra of IL-1 and IL-2 were recorded in a 300 MHz

spectrometer in solutions of CDCl3. Chemical shifts were expressedin parts per million (ppm, d) with reference to CHCl3 (d = 7.26 ppm)as an internal standard. All coupling constants (J) were absolutevalues, expressed in hertz. The description of the signals includess = singlet, d = doublet, and m = multiplet. Detailed results havebeen depicted in Text S-1A in SM.

2.2.2. Tensiometry (ST)Tensiometric measurements were taken with a calibrated tensi-

ometer of (Jencon-India) following the ring detachment technique.4–5 mL solvent of desired concentration was taken in a thermo-stated (accurate within ±0.1 K) double walled (jacketed) glasscontainer at 298 K. A stock surfactant solution of the desired con-centration (10–20 times CMC) in the desired solvent mediumwas stepwise added with a Hamilton microsyringe, stirred usinga magnetic stirrer after each addition followed by measurementof surface tension was taken after equilibration. Duplicate mea-surements were taken to check reproducibility. Further details ofthe experimental procedure can be found in our earlier reports[45,46].

2.2.3. Conductometry (cond.)A digital conductivity meter (Eutech-EcoScan-Con 5, Singapore)

was used for conductance measurements. The measurements weretaken in a constant temperature water bath, (accuracy of ±0.1 K)using a dip type cell of cell constant k = 1.0 cm�1, under constantstirring condition allowing sufficient time for equilibration [47].Details of different types of measurements can be found in TextS-1B in SM.

Table 1Influence of [IL-1] on the CMC, b , DHo

m of DTAB, SDS and CHAPS self-aggregation studied by different methods (ST-surface tension; cond-conductance; lCal-microcalorimetry) at298 K.a,b,c,d

[IL-1] DTAB SDS CHAPS

ST (Plateau) Cond (b) lCal �DHom ST (Plateau) Cond (b) lCal �DHo

m ST (Plateau) lCal �DHom

0.00 16.2 15.1 (0.71) 16.7 2.95 9.1 8.6 (0.6) 9.9 2.6 5.3 5.1 1.540.05 11.0 (30.2) 15.7 (0.67) 17.9 4.61 – – – – – – –0.10 11.2 (24.6) 17.6 3.64 4.7 (12.6) – 5.5 0.49 – – –0.20 – – – – – 3.6 (0.32) 4.7 0.66 4.8 (20.0) 4.6 0.310.30 11 (19.8) – 15.4 3.78 – – – – – – –0.50 8.3 (17.6) – – – 2.2 (4.2) – 3.8 1.00 – – –1.00 7.4 (19.0) 9.7 (0.35) 10.7 2.24 – – – – – – –2.00 3.4 (5.6) 6.75 (0.37) 7.31 2.44 1.2 (2.5) 1.5 (0.35) 1.7 2.10 3.6 (14.0) – –10.0 2.4 (4.2) 1.74 (0.42) 2.84 6.28 0.4 0.8 (0.57) 1.0 2.23 3.5 (11.1) 3.4 0.6320.0 0.7 (1.4) – 1.44 9.77 – – 0.8 2.90 – – –50.0 0.9 (4.0) – 1.05 9.63 0.3 (2.3) – 0.3 5.89 2.9 (13.5) 2.8 1.31200 – – – – 0.1 (0.4) – 0.1 9.42 – – –

a [IL-1] and CMCs (by ST, cond. and lCal.) are expressed in 10�3 M unit ; DHom are expressed in kJ mol�1.

b Average errors in CMC were ±8%; errors in enthalpy ±5%.c CMCs of pure aqueous surfactant solutions were close with earlier reports [64].d b = [1 � (post-CMC slope/pre-CMC slope)] in j vs. C plot [47,48].

I. Mukherjee et al. / Journal of Colloid and Interface Science 395 (2013) 135–144 137

2.2.4. Microcalorimetry (lcal)An Omega ITC microcalorimeter (Microcal Inc., Northampton,

USA) was used for the microcalorimetric measurements. A NeslabRTE-100 (USA) water bath was used for maintaining experimentaltemperature. Heat of dilution of the targeted surfactant solutionwas measured. The experiments were repeated to check reproduc-ibility. The measurement details and data analysis can be found inour previous reports [48] and also in the Text S-1C in SM.

2.2.5. Langmuir film balance (LB)The Surface pressure behavior of the IL-2 was measured in a

Langmuir balance, Model LB2000C – Apex Instrument Co. (India),with a vibration isolation arrangement. The trough and the barrier(composite part of the instrument) were made of Teflon. A plexi-glass box was used in the system for preventing the entry of dustparticles into the stage and the trough. The changes in the surfacepressure (p) were measured by using a film electro-balance (Sarto-rius) of resolution 0.01 mN m�1 connected with a computer. Theexperimental temperature was 298 K, and the data were taken ata lateral compression rate of 5 mm/min. Duplicate measurementswere taken to check reproducibility of the data. More informationon the instrument and its function can be found in our earlier re-ports [49,50].

2.2.6. Phase behaviorThe phase forming behaviors [45] of pseudoternary systems

comprising water/IPM/IL-2+IP (A), IL-1/IPM/IL-2+IP (B) and IL-1/IPM/IL-2+Bu (B1) were examined. For (A), a fixed amount of thesurface-active material (IL-2) with co-surfactant IP in definiteweight ratio was taken in different amounts of oil (IPM) in a setof dry test tubes with a Hamilton microsyringe to have solutionsof different strengths in them. Water in increasing quantities wasthen added to these solutions under a constant stirring condition.The test tubes were then placed in the thermostatic water bath(accuracy ±0.1 K) to note the phases obtained in them at differenttemperatures. The solutions were then allowed to settle to studytheir stabilities with time and temperature. They were afterwarddestabilized by agitation and allowed to re-equilibrate to checkreproducibility. In the case of the other two systems, (B) and(B1), IL-2 with co-surfactant IP and Bu in a fixed weight ratiowas taken in different amounts of IL-1 (polar liquid) in a set ofdry test tubes to have solutions of different strengths in them asin the previous protocol. IPM (oil) was then added to the mixtures.

The weight percent compositions of the components in (A), (B) and(B1) were calculated, and the results were plotted on triangular co-ordinates to construct the pseudo-ternary phase diagrams.

2.2.7. Dilution experimentIn this experiment [51,52], a desired amount of IL-2 was taken

in a dry test tube followed by addition of IL-1 and oil into it in spe-cific amounts governed by the phase diagram to get a desiredx([IL-1]/[IL-2]). The tubes were then placed in a thermo-statedwater bath (accuracy ±0.1 K), stirred constantly, and kept well cov-ered to prevent loss by evaporation. Then alkanol was slowlyadded to the initial viscous and turbid solution by a Hamiltonmicrosyringe until the solution became just clear, allowing suffi-cient time for equilibration. The volume of alkanols (IP or Bu) re-quired at that point was noted. A known but a small amount ofoil was then added to the freshly formed clear solution to destabi-lize it. The reappeared turbidity was then made to disappear byadding a known volume of either IP or Bu. This procedure was re-peated several times. The entire protocol was followed at three dif-ferent temperatures 298, 303, and 313 K. The experiments wererepeated twice to check reproducibility.

2.2.8. Dynamic light scattering (DLS)DLS measurements [53] were taken in a Malvern Zetasizer

Nano-S (Malvern Instruments, UK) equipped with He–Ne laser(633 nm, 4 mW) at 298 K at a fixed angle 173� (back scatteringdetection). 3.5 mL cuvettes were used for the study. All solutions(microemulsion systems of different compositions of A and B) werefiltered 2–3 times through 0.45 lm pore size cellulose acetate filterfor removing extraneous particles. Measurements were repeatedthrice and the average values of droplet diameter (d), polydisper-sity index (pdi, the ratio of the standard deviation in d and theaverage d), and the diffusion coefficient of the dispersed droplets.Safavi et al. [54] and Rojas et al. [55] recently reported DLS studyresults on IL based microemulsion systems.

2.2.9. Thermal stabilityTemperature sensitivity of the microemulsion systems (A) and

(B) was studied following our earlier protocols in the study ofclouding of amphiphiles and polymers [56,57]. Microemulsionsolution was taken in a 5 mL stoppered test tube and then placedin a heating mantle for controlled heating with constant stirring.Visual changes in the system during heating were noted. The

138 I. Mukherjee et al. / Journal of Colloid and Interface Science 395 (2013) 135–144

reverse process, that is, cooling was also studied. One repeat mea-surement was taken to check reproducibility.

2.2.10. Anti-cancer activityAnti-cancer test for the ILs was done by the traditional proce-

dure, Trypan Blue Test by following the standard protocol [58].An American Optical 110 Phase Contrast Microscope – (Dark PhasePlan Achromatic Objectives 10�, 40�, 100�) – was used in thestudy. Cell counting was done in a Hemocytometer. A detailed pro-cedure has been given in the Text S-1D in SM. All experimentswere performed in compliance with the relevant laws, and institu-tional guidelines and approval.

3. Results and discussion

3.1. Solution behavior of IL-1 and IL-2

3.1.1. Self-assembly formationIL-1 is highly soluble in water, but IL-2 is very weakly soluble.

Former was found to have good surface activity but did not formmicelle in solution. A gradual decrease in c was observed up to31.4 mN m�1 at [IL-1] = 2.2 M (83% by mass) without showing aCMC (Fig. 1a). Since tensiometry is the most effective method todetect CMC, we considered that our conclusion was correct. In thisregard, IL-1 was comparable with tetraalkyl salts viz., tetramethyland tetraethylammonium bromides studied by us earlier [56]. Ten-siometric measurements (Fig. 1b) evidenced a CMC of IL-2 at0.03 � 10�3 M. TX-100 produced a CMC of 0.22 � 10�3 M, seven-fold larger than IL-2. The CMC of Tween-20, 40, and 60 are 0.05,0.023, and 0.021 � 10�3 M, respectively, and that of Brij-35 and70 are 0.06 and 0.02 � 10�3 M, respectively. The CMC of IL-2compared well with Tweens and Brijs. Phosphorous based non ILsurfactants dodecyl-, tetradecyl-, and hexadecyltriphenylphospho-nium bromides have shown larger CMC values of 2.53, 0.69, and0.095 � 10�3 M, respectively, at 303 K [57]. The differences in thehead groups and the counter ions are considered the reasons forthese variations. The low CMC of IL-2 was caused by large hydro-phobicity produced by the attached three hexyl and one tetradecylchains to the phosphonium cation of the molecule. It is evidentfrom Fig. 1a and b that at 0.1 � 10�3 M, IL-1 reduced the surfacetension (c) of water to 68 mN m�1, whereas IL-2 reduced it to35 mN m�1 at 298 K. At this concentration, surface tension ofTX-100 (cTX-100) became 45 mN m�1. In the surface activity scale,IL-2 was well up.

Surfactants in combination can form mixed micelles with CMCsdifferent from that of the pure components [59]. In practical,situation mixed surfactants perform better than their individualcomponents. We have herein studied binary mixtures of IL-2 andTX-100 (Fig. 1b and Fig. S-1B in the main text, and SM, respec-

Fig. 1. (a) Tensiometric profile of IL-1 in aqueous medium at 298 K. Inset: tensiometric pc � log C profiles of IL-2+TX-100 binary mixtures at mole ratios 1:0, 1:1, 1:2, 1:4 and 0:

tively); the latter is often used in combination with othersurfactants [60,61]. In Table S1 (in SM),, tensiometric and conduc-tometric results of the formed mixed systems are presented. Sys-tematic increase in CMC of the mixtures with increasingproportions of TX-100 was observed. Pure IL-2 produced threebreaks in the conductometric plot, so also its mixtures with TX-100. IL-2 produced breaks at 0.004, 0.16, and 1.07 � 10�3 M. Ofthe three breaks, the first at 0.004 � 10�3 M (panel B of Fig. S-1Bin SM) was common to all mixed compositions with TX-100 ofmole ratios 1:0, 1:1, 1:2, 1:4, 1:6, 2:1, and 4:1; the positions forthe second and third breaks were composition dependent (panelA of Fig. S-1B in SM). None of these breaks matched with theCMC points registered in tensiometry (Fig. 1b). The conductancebehavior revealed variable internal interactions in addition toself-association of the mixed species.

Tensiometric results of CMC of mixed micelles were testedagainst Clint’s [59] ideal mixed micelle formation model (Eq. (1)).

Clint equation :1

CMCmix¼Xn

i¼1

Xi

CMCi

� �ð1Þ

where CMCi is the CMC of the ith species with mole fraction Xi in themixture, n is the number of amphiphile species in the mixture, andCMCmix is the expected value for the mixed system. Experimentallyobserved CMCmix did not satisfy Eq. (1); mixing was non-ideal. Po-sitive deviations were observed (Fig. S-1A in SM), suggesting a mu-tual antagonistic interaction between IL-2 and TX-100 in theirmixed forms.

Above results were also tested in terms of regular solution the-ory of Rubingh [61]. This model failed to derive information on theobserved non-ideality of the mixed system. The mixed system hasshown exceptionality in their solution behavior.

3.1.2. Effect of IL-1 on amphiphile aggregationSalts are known to influence amphiphile self-aggregation. IL-1 is

a highly water soluble non-micelle forming surface-active salt. ItsCMC influencing effect on the surfactants TX-100 (nonionic), DTAB(cationic), SDS (anionic), and CHAPS (zwitterionic) was studied bydifferent methods (see illustrations Fig.2a, b in the main text andFig. S-2A in SM).

The CMC of TX-100 in water increased from 0.22 � 10�3 M to0.5 � 10�3 M in the presence of 0.07 M IL-1 at 298 K. A representa-tive plot is exemplified in the inset of Fig. 1a. The triisobutylmeth-ylphosphonium cation of IL-1 interacted with the polarpolyoxyethylene moiety of TX-100 to resist its micellization. Onthe contrary, IL-1 decreased the CMCs of SDS, DTAB, and CHAPS(Table 1). It is known that salts decrease the CMC of surfactants,particularly of ionic surfactants. We have observed larger CMCreducing effect of IL-1 than common electrolytes: in presence of0.05 M IL-1, the CMC of DTAB, SDS, and CHAPS were decreased

rofile of TX-100 with 0.07 M IL-1 at 298 K. (co-ordinates: same as in main plot). (b).1 at 298 K; (co-ordinates are indicated by arrow heads).

Fig. 2. (a) IL-1 influenced c � log C profiles of SDS, DTAB, and CHAPS in aqueous medium at 298 K, with broad arrow heads indicating starts of plateau points. [IL-1]used = 0.01 and 0.05 M. Co-ordinates indicated by narrow arrowheads. (b) Enthalpograms of SDS, DTAB and CHAPS in aqueous medium with [IL-1] and NaBr at 298 K. [IL-1]used = 0.02 M and 0.05 M; [NaBr] = 0.05 M; Co-ordinates indicated by arrowheads.

I. Mukherjee et al. / Journal of Colloid and Interface Science 395 (2013) 135–144 139

by 1/18, 1/30, and 1/2-fold, respectively. Effect on SDS was thehighest. Effects of IL-1, NaBr, and NaCl on the CMCs are comparedin Table 1 and Table S2 in the main text and SM, respectively. Themuch reduced CMC by IL-1, compared to normal salt, resulted fromthe uncommon counter ion environments for both SDS and DTAB.Behera and Pandey [23] reported a concentration dependent dualCMC influencing effect on SDS by the hydrophilic ionic liquid,1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF4]). Itdecreased the CMC in the concentration range, 0 < [bmim][BF4]6 2 wt%, thereafter, the CMC increased with increasing concentra-tion of [bmim][BF4] studied up to 30 wt%. At low and high concen-trations, the IL behaved like co-surfactant and co-solvent,respectively. CMC reduction phenomenon of [bmim][BF4] on SDSat 62 wt.% matched with our results. DTA+ interacted with tosylateanion to reduce its CMC. According to our findings, zwitterionicsurfactant CHAPS balanced the influences of both cation and anionof IL-1 to manifest a moderate effect. Weak charge interaction be-tween the cationic part of IL-1 and the anionic part of CHAPS andfavorable orientation of the triisobutyl groups of the former alongthe tails of the latter favored formation of micelle with reducedCMC. Beyond CMC, all the surfactants produced a rise in c endinginto formation of a plateau. The micelle-monomer equilibrium be-tween the bulk and the interface changed with altered micellemorphology causing an increase in c that finally leveled out toform the plateau. The ratios between the plateau point and theCMC for DTAB, SDS, and CHAPS were on the average 1.8, 2.7, and3.9, respectively, excepting at [IL-1] = 0.05 M for SDS with a higherratio of 7.7. The plateau formation occurred sequentially at higherconcentration of the studied surfactants. In the c versus log [surfac-tant] plot (Fig. 2a) beyond CMC, formation of plateaus correspond-ing to [IL-1] = 0.05 M is marked with broad arrow heads.

Concentrations of plateau positions at different [IL] are shownwith CMC in first brackets in Table 1. We had made a preliminaryattempt to test the aggregation behavior of IL-2 in aqueoussolution of IL-1, that is, solution behavior of one IL into another.A low concentration (3 � 10�3 M) of IL-1 was found to produce astriking effect on IL-2. In the c versus log [IL-2] dependence, aCMC like inflection was observed at a very low concentration of0.0001 � 10�3 M at 298 K (whereas in aqueous medium withoutIL-1, its CMC was found to be (0.03 � 10�3 M, as stated above).There were reports of CMC decrease on mutual interaction be-tween two ionic liquids [62]; interaction in the IL-2/IL-1 combina-tion herein found was conspicuous (Fig. S-2C in SM).

Magnitudes of CMCs by calorimetry [63,64] for the ionic surfac-tants were found to be higher (specifically for the cationic DTAB);the zwitterionic CHAPS produced nearly equivalent CMCs both byST andl Cal. CMC values of DTAB by tensiometry (CMCST) wereroughly 50% lower than the CMC by conductometry or microcalori-metry (CMCcond/lcal); conductometric CMCs were closer to micro-

calorimetric values. These differences are graphically presentedin Fig. S-2B in SM. log CMCST maintained almost a constant differ-ence (�3.6 unit) with log CMClcal and log CMCcond.

For the studied systems with increasing [IL-1], an initial declinefollowed by an increase in the exothermic magnitude of DHo

m (stan-dard enthalpy of micellization of SDS, DTAB and CHAPS) was ob-served (Table 1). A comparison of the effects of NaBr and NaCl onthe DHo

m of DTAB and SDS, respectively, has been made inTable S2 in SM; magnitude of the exothermicDHo

m increased withincreasing concentrations of NaCl and NaBr. Co-surfactant propertyof the amphiphilic salt (IL-1) made an energetic difference from thenormal salts.

3.2. Interfacial behavior of IL-2

3.2.1. Gibbs monolayer (GM)Strongly surface-active IL-2 yielded a positive surface excess C

at the air/water interface. The Gibbs adsorption equation was usedto determine Cmax from the c vs. log [IL-2] plot in the usual way[53,72],

Gibbs equation : Cmax ¼ �1

2:303iRTLt

½Surf�!CMC

dcd log½IL-2� ð2Þ

where i (=2) is the number of species formed by dissociation of IL-2in solution, R is the universal gas constant, and T is the absolutetemperature. Minimum head group area (Amin) of the amphiphile(IL-2) at the air/water interface was obtained from the followingrelation [65],

Amin ¼ð1018Þ

NACmaxnm2 molecule�1 ð3Þ

where NA is the Avogadro number. Cmax, and Amin values for IL-2were 8.25 � 10�7 mol m�2 and 2.01 nm2 molecule�1, respectively,at 298 K determined from c vs. log [IL-2] curve at the CMC point.The Amin value of IL-2 was larger than the cationic surfactants alkyl-trimethylammonium bromides (0.43–0.46 nm2 molecule�1 on theaverage [66–68]). From the molecular structure (Scheme 1), the lar-ger value of Amin for the amphiphilic cation of IL-2 was expected. At298 K, Amin of the carbazole tailed ILs, for example, [carbazole Cn

mim][Br] with n = 6, 10 and 12 were reported [69] to be 2.11,1.91, 1.93 nm2 molecule�1, respectively, which were close to our re-sult. Reported [70] area per molecule of dodecyl-, tetradecyl-, andhexadecyltriphenylphosphonim bromides (0.74, 0.88, and1.63 nm2 molecule�1, respectively) were fairly lower than that ofIL-2. Hydrophobic hydration of the three hexyl chains in the headgroup of IL-2 made the Amin higher.

140 I. Mukherjee et al. / Journal of Colloid and Interface Science 395 (2013) 135–144

3.2.2. Langmuir monolayer (LM).Very low water soluble IL-2 conveniently formed a Langmuir

monolayer [49,50] at the air/water interface, which was studiedwith a Langmuir balance. 5 lL of 1.79 mg mL�1 of IL-2 in CHCl3

was added on the water surface (sub-phase) in a Langmuir troughof 420 cm2 area. Under static condition (no barrier movement),surface pressure p slowly increased and reached equilibrium after3 h (trend depicted in Fig. 3a, curve 1). The determination of pres-sure – area isotherm appeared inconvenient. On a trial basis, thepressure area (p � A) isotherm measurement was taken soon(within 5 min) after the addition of the sample to the sub-phase(Fig. 3b). The steeper portion of the compressed monolayer whenextrapolated produced a head group area of 3.7 nm2 molecule�1.After the run, the compressed monolayer evidenced stability; anexpansion run was then taken. It displayed hysteresis; the steeppart on extrapolation produced the head group area of 2.7 nm2 -molecule�1. In another experiment, after a prolonged (overnight)stay of the added IL-2 on the sub-phase the system witnessed sta-bilization with zero surface pressure. Its compression then pro-duced an isotherm (Fig. 3a, curve 2), which yielded an identicalhead group area of 2.7 nm2 molecule�1. The compressibility modu-lus of the isotherms (C�1

s ¼ AdpdA ) is shown in the insets of Fig.3. The

maxima fell in the range of liquid expanded state (12.5–50 mN m�1) [50]. The Gibbs monolayer (GM) (discussed above)produced a head group area of 2.01 nm2 molecule�1 at CMC. TheLM yielded a head group area 35% greater than that of GM. Interfa-cial behavior of insoluble LM of ILs requires more exploration.

3.3. IL-1 and IL-2 based microemulsions

3.3.1. Phase behaviorThe pseudoternary phase diagrams of systems (A) and (B) are

depicted in Fig.4a and b and in Figs. S-3A and 3B in SM, respec-tively. In Fig. 4a, clear and turbid zones are marked for IL-2: IP ra-tios (wt/wt) 1:1, 1:2 and 1:4. The clear zones were much smallerthan the turbid (or biphasic) zones; former only moderately in-creased with an increasing proportion of IP in the amphiphile mix-ture. Phase forming behavior was practically independent oftemperature in the range of 298–323 K (Fig. S-3A in SM). Pointwise (wt.%) phase compositions of IL-2: IP as 1:4 in the orderwater/IPM/(IL-2+IP) are herein presented for a ready understand-ing of the trend. From IPM corner to the IPM-(IL-2+IP) baselinethe compositions were sequentially as follows: 1.2/82.6/16.2, 2.1/75.7/22.3, 3.2/68.2/28.6, 5.3/54.6/40.1, 10.2/36.3/53.7, 17.9/20.8/61.3, 20.9/15.2/63.9, 26.0/10.7/63.2, 26.2/6.8/66.9, 30.0/2.3/67.7,34.5/1.7/63.8, 28.8/1.2/69.9, and 35.9/0.4/63.7. The situation forthe system (B) was radically different. A very large clear zone

Fig. 3. p � A isotherm of LM of IL-2 at the air/water interface at 298 K (a). 1: incrememeasured after a day of addition on the interface. Inset: display of compression modulus oa half cycle documented. Inset: compression modulus of the formed LM.

was observed both for IP and Bu as co-surfactants in combinationwith IL-1 as the polar phase and IL-2 as the surfactant. DecreasingIL-2: IP ratio (from 2:1 to 1:4 wt/wt) caused a perceptible increasein the clear zone (Fig. 4b).

IP and Bu were at par in their efficacy (Fig. S-3B in SM). Thus, Bis an interesting system with two ILs (one polar, IL-1 and another asurfactant, IL-2 with a pharmaceutically important useful co-sur-factant IP and the nontoxic oil IPM yielded very large clear zones,not frequently found in practice). It thus has a potential for a stablecost effective and useful microheterogeneous system. As above, atIL-2:IP as 1:4 (wt/wt) for system B the sequence of point to pointwt.% composition from IPM to IL corner as IL-1/IPM/(IL-2+IP), wereas follows: 1.5/94.0/4.5, 3.5/89.3/7.2, 11.7/76.1/12.2, 36.9/47.8/15.3, 63.7/19.8/16.5, 77.6/8.9/13.4, 94.1/1.7/4.2. Percent areas ofthe clear zones [71] of the studied systems are presented inTable S3 (in SM) with the areas of other recently reported systems[28,54,55,72]; herein, studied system (B) tops the list. System (B)became clear viscous liquid with increasing IL-1 after a thresholdwt.% composition of 72.5/5.7/21.8 as IL-1/IPM/(IL-2+IP) for all thestudied ratios. A change of temperature in the range of 298–323 K had a minor effect on the system phase behavior. Severalphysicochemical properties of the formed microemulsions werealso studied: these were (1) conductance behavior, (2) interfacialcomposition and stability, (3) droplet dimension, and (4) thermalstability. They are chronologically presented and discussed below.

3.3.2. Temperature induced conductance of IL-1 in IPM (IL/IPM)microemulsion

Temperature dependent conductance profile (r � T) of a typicalmicroemulsion system at x ([IL-1]/IL-2) = 5 (an arbitrary chosenratio) is exemplified in Fig. 5.

Both heating and cooling courses evidenced narrow hysteresisall through. A distinct break in the forward conductance coursewas observed at 293 K which shifted to a lower temperature(289 K) in the reverse course. Replacement of Bu in place of IPslightly increased the break from 293 to 296 K; reverse (cooling)course was not studied. The r � T profiles were not percolatingtype [48,51,73]. r, increased with increasing thermal energy; thebreaks with lower slopes meant barrier or hindrance to the ionflow in the oil continuum wherein the dispersed charged entitiesmigrated with a specific mechanism under the applied electricfield.

3.3.3. Interfacial and composition stabilityUnderstanding of the interfacial co-surfactant and surfactant

composition as well as the distribution of the co-surfactant be-tween the interface and the oil can quantitatively account for the

nt in p after the addition of IL-2 on the interface with a fixed barrier. 2: Isothermf the formed LM (b). Measured 5 min after addition of IL-2 on the interface. One and

Fig. 4. (a) Pseudoternary phase diagram of water/IPM/IL-2+IP system at IL-2:IP ratios (wt/wt) 1:1, 1:2 and 1:4 at 298 K. (b). Pseudoternary phase diagram of IL-1/IPM/IL-2+IPsystem at IL-2:IP ratios (wt/wt) 2:1, 1:1 and 1:4 at 298 K.

Fig. 5. Conductance – temperature profiles (heating and cooling) of IL-1/IPM/IL-2+IP at x = 5. Inset: same with IL-2+Bu (only heating profile). Co-ordinates same asmain plot.

I. Mukherjee et al. / Journal of Colloid and Interface Science 395 (2013) 135–144 141

thermodynamic stability of microemulsion. Dilution experiment(described in the Section. 2) has been successfully employed forunderstanding the above [51,52,74]. The two systems (B) and(B1) described in Section. 2.2.6., were tested also at x ([polar li-quid]/[surfactant]) = 5. In a polar liquid/oil microemulsion system,relations presented as Text S3 in SM can be formulated [52,75].

We rewrite Eq. (7) from the Text S-3 in SM here as Eq. (4) toshow its use.

Thus;na ¼ 2kno þ nia ð4Þ

Fig. 6. (a) na/ns vs. no/ns profiles for the system IL-1/IPM/IL-2+IP at x = 5 Main plot: 298for the system IL-1/IPM/IL-2+Bu at x = 5 main plot: 298 K; Inset: 303 K (co-ordinates sa

Dividing both sides of Eq. (4) by ns the form changes to

na

ns¼ 2kno

nsþ ni

a

nsð5Þ

The distribution constant Kd for the equilibrium of IP betweenthe interface and the oil is given by the relation,

Kd ¼ Xia=Xo

a ¼ni

a=ðnia þ nsÞ

noa=ðno

a þ noÞ¼ Ið1þ S=2Þ

S=2ð1þ IÞ ð6Þ

where I ¼ nia=ns, and slope, S ¼ 2k ¼ 2ðn0

a=n0Þ;)S=2 ¼ n0a=n0

Use of the linear Eq. (5) can determine nia=ns and k from the

intercept and, the ½ (slope), respectively. The known values of nia

and noa can be used to calculate the distribution constant, Kd (Eq.

(6)) to get the Standard Gibbs free energy of transfer(DGo

t ¼ �RT ln Kd) of IP from the oil phase to the interface to makea stable nano-dispersion of IL-1 in IPM.

Results of the systems B and B1 at x = 5 are exemplified inFig. 6. Eq. (5) was applicable; the slope and the intercept valuesare presented in Table 2 with the corresponding Kd values that de-creased with temperature. The transfer enthalpy DHo

t was exother-mic as found in water based microemulsions [46,48]. DHo

t , and DSot

(transfer entropy) values are shown in Table 2. Relations used aregiven in the footnote. DGo

t (B) > DGot (B1) (the second process was

more spontaneous); DHot (B) was �1/2 DHo

t (B1). The negative en-tropy values of B1 were much higher than (B). Exothermic dropletformation stabilized the system with large negative entropychange. In conventional w/o microemulsions with hydrocarbonoils and synthetic detergents, both endothermic and exothermicDHo

t associated with positive and negativeDSot , respectively, were

reported [46,52].

K; Inset: 303 and 313 K (Co-ordinates same as main plot.) (b). na/ns vs. no/ns profilesme as main plot.).

Table 2Results of dilution experiments with IL-1/IPM/IL-2: IP or Bu microemulsion system at x = 5 at 298, 303 and 313 K.a

(B) IL-1/IPM/IL-2:IP(1:1 wt/wt) (B1) IL-1/IPM/IL-2:Bu(1:1 wt/wt)

T/K 298 303 313 298 303I 2.77 1.28 0.86 8.8 4.91S 0.61 0.69 0.86 0.17 0.27Kd 3.11 2.17 1.54 11.5 6.98�DGo

t kJmol�1 2.81 1.95 1.12 6.05 4.89

�DHot (kJmol�1)a 35.1 35.1 35.1 74.9 74.9

�DSot (JK�1 mol�1)a 108.4 109.4 108.6 231.0 231.1

For system B: DHot ¼ ½@ðDGo

t =TÞ=@ð1=TÞ�p and for system B1: DHot ¼ R lnðKdð1Þ=Kdð2ÞÞ � ðT1T2=ðT2 � T1ÞÞ. 1 and 2 refer to temperatures 298 and 303 K, respectively. For both the

system B and B1 DSot ¼ ðDHo

t � DGot Þ=T . Errors in DGo

t , DHot and DSo

t were ±3%, ±6%, and ±10%, respectively.a The relations used for evaluating DHo

t and DSot were as above.

142 I. Mukherjee et al. / Journal of Colloid and Interface Science 395 (2013) 135–144

Herein, reported energetic parameters are with reference tox = 5. Results at other x would vary, since dispersed droplets’ sizedepends on system composition. For a fixed surfactant and co-sur-factant concentration at a fixed mass ratio in the continuous oilphase, progressive addition of polar liquid should increase dropletsize with a varied surface area with an altered population of surfac-tant and co-surfactant molecules to make changes in the distribu-tion coefficient Kd . The energetic parameters would consequentlychange [51]. ‘‘x’’ dependent energetic studies with ILs as compo-nents would be thus interesting and informative for producing auseful microemulsion forming solution.

3.3.4. Droplet dimensionDroplet dimensions in the prepared systems (A) and (B) were

determined by DLS method. Overall, diameter (d) and polydisper-sity index (pdi) values of the dispersed water droplets in the IL-2stabilized system (A) are presented in the top part of Table 3. Ata fixed IL-2: IP wt ratio of 1:1, droplet size only moderately in-creased with increasing proportion of water; average dropletdimensions ranged within 2.7–4.0 nm. Their x values ranged be-tween 2 and 4. The species were like reverse micelles (RM)although their water content was not trace, a requirement forthe formation of RM of surfactants in oil. They thus may be consid-ered to fall in the border line between RM and microemulsion. Thepdi values obtained were mostly large; the systems were apprecia-bly polydisperse. At IL-2: IP wt ratio 1:2, higher proportion ofwater made droplet sizes three times larger with lower pdi. For B(bottom section of Table 3) at IL-2: IP weight ratio 1:1, dropletdiameters were all larger than A. The last one at IL-2: IP (wt/wt) = 2: 1 produced large dispersion size. Composition wise, itwas a bi-continuous type system. System (B) produced moderatelypolydisperse dispersions with small changes in droplet diameters.Average droplet size dependent intensity profiles are depicted in

Table 3DLS results of dave and pdi of microemulsion systems: A-water/IPM/IL-2+IP and B-IL-1/IPM/IL-2+IP at varied compositions at 298 K.

daave (nm) pdib

System A wt.% composition of water/IPM/IL-2+IP (IL-2: IP)1.3/66.3/32.4 (1:1) 2.7 0.92.6/49.6/47.7 (1:1) 3.4 0.93.2/36.6/60.2 (1:1) 3.4 0.84.7/28.7/66.6 (1:1) 4.0 0.812.8/20.5/66.6 (1:2) 11.7 0.1

System B wt.% composition of IL-1/IPM/IL-2+IP (IL-2: IP)1.9/87.1/10.9 (1:1) 12.0 0.53.6/77.9/18.4 (1:1) 12.8 0.59.5/60.0/30.4 (1:1) 12.6 0.330.9/30.8/38.2(1:1) 16.9 0.435.9/35.8/28.2 (2:1) 217 0.3

a Average of five readings reported. The standard deviations in dave were ±4% in A,and ±14% in B.

b pdi, the ratio of the standard deviation in ‘‘d’’ and the average ‘‘d’’

Fig. S-4A in SM. In this context the report of Rojas et al. [28] maybe cited, where droplet enlargement of the microemulsion system(comprising 1-ethyl-3-methylimidazolium ethylsulfate[emim][EtOSO3] as (polar phase)/toluene (oil)/1-butyl-3-methyl-imidazolium octylsulfate[bmim][OctOSO3] as (surfactant) at298 K) was observed with increase in the polar phase from 6 to21 wt.% in partial agreement with our observation.

Thermal effect on the diameter of IL-2 and IP stabilized waterand IL-1 dispersions in (A) and (B) are presented in Table S4 inSM. Both dispersions produced decreased size with increased tem-perature. For a temperature increase of 25 K, droplet size of system(A) decreased by 65% while that of system (B) decreased by 36%.The pdi values for the first set were all large which for the secondset were much lower (Table S4 in SM). On cooling sizes of disper-sion (A) and (B) increased fairly and moderately. At the lower endof temperature (298 and 303 K), dispersion (B) produced two cat-egories of species, one much smaller than the other. Their temper-ature dependent size distributions are exemplified in Fig. S-4B inSM.

Concentrations of the two systems used in DLS study were notlow; the measured diameters were thus influenced by inter-drop-let interaction. Results of systems >5 wt.% were thus consideredapparent.

3.3.5. Thermal stabilityTemperature sensitivity of phase behavior of system (A) was

studied in the range of 298–323 K by a 5 K increase in each step.Almost overlapping results (Fig. S-3A in SM) were observed to con-clude that in the studied range system (A) was on the whole tem-perature insensitive. The thermal stability of the variouscompositions of (A) and (B) was also examined. At higher temper-ature (>353 K) biphasic solutions of varied consistencies were ob-tained. Details are presented in the Text S-2A in SM which isrequired for uses of IL derived microemulsions in practice.

4. Anti-cancer activity.

Anti-cancer property of IL-1 is distinctly evidenced from the re-sults on the Sarcoma 180 cell line, presented in Table S5 in SM.5 mg/mL of IL-1 in 0.9 g% NaCl solution (physical saline solution)caused 100% killing of Sarcoma-180 (1 � 105 cells/incubation mix-ture) cell line in 1 h. Half dilution of the aforesaid IL-1 solutioncauses a 19% reduction in killing efficacy. Five and tenfold dilutionresulted 47% and 78% reduction of killing efficacy, respectively. IL-2on the other hand has less anti-cancer activity than IL-1. 10 mg/mLIL-2 took almost 2 h to kill 89% of Sarcoma-180 cells, and 1 h for58% of the cells. 10 mg/mL was equivalent to 1.0 � 10�3 M IL-2solution, which was quite higher than its CMC (0.03 � 10�3 M).Both IL-1 and IL-2 molecules have large structural differences;the latter has a poorer aqueous solubility than the former. IL-1did not aggregate to form micelle; its entry through the cell

I. Mukherjee et al. / Journal of Colloid and Interface Science 395 (2013) 135–144 143

membrane was easier compared to IL-2 which supplied monomersof concentration of 0.03 � 10�3 M, the rest 0.97 � 10�3 M re-mained out as bigger assembled and non-penetrable units. Frompenetrating proficiency IL-1 effectively supplied more moleculesthan IL-2 to augment better anti-cancer activity. Thus, the activityshown was virtually equivalent to its minimum inhibitory concen-tration (MIC) that Mandal et al. [76] reported for the antibacterialhydantoin drug.

Cancer is an immunosuppressive disease; hence cancer patientbecomes much susceptible to secondary infections (like bacterialinfections). Herein, studied ILs showed anti-cancer activity andearlier study showed their antibacterial activities [44] against bothgram-positive and gram- negative strains. Thus, our experimentsevidenced a broad prospect of the therapeutic applications of thetwo ILs herein studied. More investigations in both in vitro andin vivo conditions are required.

5. Conclusion

Of the two RTILs, IL-1, and IL-2, the latter is more surface activethan the former and can form micelles in aqueous medium whichthe former cannot. The water soluble IL-1 forms Gibbs monolayersat the air/water interface whereas the sparingly soluble IL-2 formsboth Gibbs and Langmuir monolayers. The latter shows liquid ex-panded type p � A isotherms like lipids in general [49,50] withappreciable hysteresis. IL-2 has a CMC of 0.03 � 10�3 M, eightfoldsmaller than the CMC of TX-100; in combination, they form mixedmicelles with mutual antagonistic interaction deviating from therule of ideal mixing of Clint as well as not in conformity with reg-ular solution theory of Rubingh [61]. IL-1 exhibits a greater CMCreduction of SDS and DTAB than NaCl and NaBr. By its influence,along with CMC both b and the magnitude ofDHo

m decreased.The mixed pseudoternary systems water/IPM/IL-2+IP and IL-1/

IPM/IL-2+IP at different IL-2: IP mass ratios and temperatures aredifferent: water-free systems produce very large single phasemicroemulsion zones, rarely found in practice. The favorable pres-ence of (IL-2+IP) at the IL-1/IPM interface than at the water/IPMinterface makes easier dispersion of IL-1 in IPM than water inIPM. This striking observation brings promises for more trials withother IL-1 like ILs in formulating prospective microemulsion sys-tems for applications. Between the systems B 1 and B, energeticallythe former shows greater spontaneity, greater exothermicity, andlarger negative entropy than the latter. The droplets are on thewhole uniform in dimensions at 1:1 weight ratio of IL-2: IP; atother ratios 1:2 and 2:1, the size increase is large. The IL-1/IPM dis-persions are less polydisperse than water/IPM. In phase formingbehavior, IL-1 is a better option than water in producing exception-ally larger monophasic zone. The formed dispersions are tempera-ture insensitive; on heating at or above the boiling point of IP(353 K) they become unstable and biphasic. Good temperature sta-bility up to 353 K suggests numerous applications of these micro-emulsion systems. IL-2 is weakly toxic material [77], it may thushave prospect in pharmaceutical preparations for topical applica-tions. IL-1 has good anti-cancer activity compared to IL-2. Theiranti-bacterial property has been already reported [44], and so theyhave prospects for therapeutic uses.

Acknowledgments

I.M. thanks Centre for Surface Science, Department of Chemis-try, Jadavpur University for laboratory facilities, Prof. K.P.D. andV.B. of Bose Institute DLS measurements, A.C. of I.A.C.S, Kolkata,for examining anti-cancer activity. Support from the INSA toS.P.M. is thankfully acknowledged. S.M. and B.N. acknowledge CSIRand UGC, respectively, for financial support.

Appendix A. Supplementary materials

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.jcis.2012.11.064.

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