Mixed micelles of cationic surfactants and sodium cholate in ... - NOPR

Indian Journal of Biochemistry & Biophysics Vol. 41, April-June 2004, pp. 107-112

Mixed micelles of cationic surfactants and sodium cholate in water Dharmesh Varade, Vijay Patel, Anita Bahadur, Pratap Bahadur* and Martin Swanson Vethamuthu† Department of Chemistry, South Gujarat University, Surat 395 007, India †

Unilever Research US. Inc., Edgewater Laboratories, New Jersey 07020, USA Received 5 May 2003; revised 29 April 2004

Critical micelle concentrations (CMCs) of cationic surfactant (alkyltrimethylammonium bromides, CnTABr, where n = 10, 12, 14, 16 and 18), and a bile salt sodium cholate (NaC) were determined from surface tension, conductance and dye solubilization methods, while of their equimolar mixtures from surface tension and dye solubilization methods. The interaction parameter (β) obtained from analysis of data, using Rubingh’s theory showed strong interaction between NaC and cationic surfactant. Time-resolved fluorescence-quenching results revealed small-sized mixed spherical micelle with aggregation number much less than micelles of cationic surfactant. Keywords: Bile salts, cationic surfactants, mixed micelles, interaction parameter.

Bile salts are typical anionic bioamphiphiles, which participate in many physiological processes1. They show unique interfacial activity and aggregation pattern in dilute solutions in water, but they do not form liquid crystalline phases in concentrated solutions1-2. However, mixtures of bile salts with other amphiphiles form mixed micelles3-6 and liquid crystalline phases1,7. The most extensively studied system has been the bile salt-lecithin-water mainly, due to its biological importance. Some studies on mixtures of bile salts and cationic surfactants have also been reported8,9. Mixed surfactants are often used to improve performance, as the mixtures are superior to a single surfactant due to the interaction between two differently charged surface-active species10-12. Earlier, aqueous mixtures of an anionic and a cationic surfactant have been found to exhibit fundamentally different properties, than the corresponding solutions of pure ionic surfactant or mixtures of an ionic and a non-ionic surfactant13-17. Strong interaction due to electrostatic forces has been shown to be present in mixtures of anionic and cationic surfactants, but oppositely charged surfactant systems often limit their use due to the formation of insoluble complex10. Interestingly, mixed surfactant systems comprising _________ *Corresponding author Fax: (0261) 2256012 Tel: (0261) 2258384 E-mail: [email protected]

sodium cholate (NaC) and alkyltrimethylammonium bromide do not form precipitate/coacervate8,9 even at equimolar concentrations. This behaviour offers a possibility to study oppositely charged surfactant systems, without the complication of phase separation. Mixed micelles are often examined by interaction parameter, β, obtained by using a simple model for non-ideal mixed micelles from regular solutions theory by Rubingh18. An attractive interaction gives a negative β value, while a repulsive interaction a positive value. The larger β value indicates stronger interaction between the two surfactants. In recent years, much attention has been paid to the aggregation of mixed systems containing bile and other surfactants. Most of the biological functions of bile salts are based on their ability to associate with molecules, such as cholesterol and lecithin to form mixed micellar aggregate structure. The role of bile salts as physiological surfactants is based on their micelle-forming properties8. In this paper, we report the results on mixed micelles from equimolar solutions of a bile salt NaC and different cationic surfactants alkyltrimethylammonium bromides CnTABr (n =10, 12, 14, 16 and 18). The critical micelle concentrations (CMCs) of single surfactants were determined from surface tension, conductance and dye solubilization methods, while of equimolar mixtures of cationic surfactant and NaC surfactant systems from surface tension and dye solubilization

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INDIAN J. BIOCHEM. BIOPHYS., VOL. 41, APRIL-JUNE 2004

measurements. Aggregation numbers (Nagg) of mixed micelles were determined by fluorescence quenching. Materials and Methods Cationic surfactants decyltrimethylammonium bromide (C10TABr), dodecyltrimethylammonium bromide (C12TABr), tetradecyltrimethyl-ammonium bromide (C14TABr), hexadecyltrimethylammonium bromide (C16TABr), and octadecyltrimethylammonium bromide (C18TABr) were highly pure samples obtained from Lancaster, England. Sodium cholate (NaC) was from Sigma Chemical Co, USA and was used without purification. A water insoluble dye orange-OT (1-o-tolyl-azo-2-naphthol) used in dye solubilization studies was prepared from o-toluidine and 2-naphthol and was used after repeated recrystallization. The probe pyrene was recrystallized twice from ethanol and dimethylbenzophenone (Aldrich), the quencher with purity >99% was used as supplied. Fresh solutions were prepared in deionized water. Methods Surface tension of surfactant solutions was determined, using drop volume technique with the help of ‘Agla’ micrometer syringe by developing the drops of solutions in air at a very slow speed for about 3 min, in all the systems NaC, cationic surfactants and their mixtures at concentrations below and above CMC. The equilibrium was established within this time and no significant change in surface tension was observed on further aging. Measurements were made at 30°C and the volume of a single drop was averaged from 5-6 measurements. The surface tension data were reproducible up to 1%. The surface tension measurements provided the CMC values (without the presence of minimum), agreeing reasonably well to those reported in literature19-21. Conductometric measurements were made with a digital conductivity meter (Phillips, India) using a dip-type cell at 30°C. All measurements were carried out in a jacketed vessel, maintained at temperature (30±0.1°C). The conductance was measured after thorough mixing and temperature equilibrium at each dilution. The errors in the conductance measurements were within ± 0.5%. Solubilization experiments were carried out by shaking an excess of dye in solution of surfactant for about 48 hr at room temperature. After attainment of equilibrium, remaining insoluble dye was separated by centrifugation and the supernatant solution

containing solubilized dye was diluted with ethyl alcohol, with final ratio of ethyl alcohol to water adjusted to 2:1. The absorbance of solution containing solubilized orange-OT at 470 nm was determined using a Bausch and Lamb spectrophotometer. For determination of aggregation number, Nagg, time-resolved fluorescence decay data were collected with single photon counting technique8. The set-up uses a mode-locked Nd-YAG laser to synchronously pump a cavity-dumped dye laser for excitation, using 4 - dicyanomethylene- 2 -methyl-6-p-dimethyl-amino styryl-4H pyran (DCM) as dye, and a potassium dihydrogen phosphate (KDP) crystal for frequency doubling. The excitation wavelength was 323 nm and pyrene monomer emission was measured at 395 nm. Results and Discussion The critical micelle concentrations (CMCs) of alkyltrimethylammonium bromides (CnTABr) and NaC determined by surface tension (γ), conductance and dye solubilization methods in water at 30°C (Table 1) show a good agreement with each other. A linear decrease in γ was observed with increase in concentrations (Fig. 1) for four cationic surfactants, beyond which no significant change was observed. This is a common behaviour shown by surfactants in solution and is used to determine their purity and CMCs22. The γ-log concentration plots also provide information about area per molecule at air-water interface. The area occupied by surfactant molecules at air-water interface at saturation monolayer was estimated, using surface tension (γ) data at temperature T and the Gibbs adsorption isotherm: Γs = −

1 ⎛ dγ ⎞ ⎜ ⎟ nRT ⎝ dlnC ⎠

... (1)

where Γs is surface excess, R is the universal gas constant, C is the concentration of surface-active compound and n is taken as 2, due to the ionic nature of surfactants. The average value of area/molecule obtained is shown in Table 1. In cationic surfactants with same head group area, but having different chain length or counterion, the area values remain essentially the same, since the area/molecule at interfaces is primarily influenced by head group size23. The CMC of NaC in water has been reported in the wide range 5-12 mM earlier by different workers1-3. The value of 9.0 mM obtained in our study can be considered in good agreement with the reported values. However, the inflexion point was not

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109

Table 1—Interfacial properties of cationic surfactants in water at 30°C Surfactants Surface tension (± 0.2)

CMC, mM Dye Conductance solubilization (± 0.1) (± 0.1)

Lit.

Counterion dissociation (α)

C10TABr

63.0

62.0

63.5

63.019

0.29

C12TABr

15.0

14.0

15.5

15.020

0.2625

C14TABr

3.50

3.40

4.01

3.8321

0.2525

C16TABr

0.80

0.80

1.04

1.0021

0.2525

C18TABr

0.34

0.30

0.39

0.3419

0.23

NaC

9.0

8.8

9.2

Fig. 1—Surface-active behaviour of alkyltrimethylammonium bromide (CnTABr) in water at 30°C [(●) C12TABr; (▲) C14TABr; (■) C16TABr; and (□) C18TABr]

distinct as observed for cationic surfactants because they do not consist of well-defined polar and nonpolar groups, but instead possess surfaces that are lipophilic and hydrophilic in nature. Conductivity measurements were performed in order to evaluate the CMC and the degree of counter ion dissociation (α). Specific conductivity is reported to be linearly correlated to the surfactant concentration in both pre-micellar and post-micellar regions24, being the slope in the pre-micellar greater than that in post-micellar region. The intersection point between two straight lines gives the CMC, while the ratio between slopes of post-micellar to that in pre-micellar regions gives α value25. The values of CMC and α are shown in Table 1. Solubilizing power is one of the most important properties of surfactants. The solubilization of water insoluble dye orange-OT in the surfactant micelles

9.0

1

--

Average Area/molecule Å2 Expt. Lit. (± 0.2)

50.7

48.514

68.2

70.01

Fig. 2—Critical micelle concentration (CMC) vs. alkyl chain length plot [(■) CnTABr; and (□) CnTABr + NaC]

was studied in order to determine the CMC of the surfactants. The amount of the dye solubilized was insignificant up to the CMC of each surfactant and thereafter, a sudden and steep rise was observed with the formation of micelles in the bulk. The CMC value for each surfactant obtained by this method (Table 1) was in good agreement with the CMC determined by surface tension and conductivity methods. The CMCs of equimolar mixtures of alkyltrimethylammonium bromides (CnTABr) with NaC were measured by surface tension and dye solubilization methods in water at 30°C. The CMCs for equimolar mixtures of C10TABr, C12TABr, C14TABr, C16TABr and C18TABr with NaC were 5.8, 0.9, 0.57, 0.17 and 0.08 mM, respectively. The CMCs for mixed surfactants were far less than the corresponding cationic surfactants and NaC due to strong synergism. Fig. 2 shows CMC values for pure CnTABr and the equimolar mixture of CnTABr +

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Fig. 3—Aggregation number (Nagg) vs. alkyl chain length (Cn) plot [(■) CnTABr; and (□) CnTABr + NaC]

NaC in water as a function of carbon chain of cationic surfactant. Such logarithmic decrease in CMC with increase in the number of carbon atoms, ‘n’, for homologous series of various surface-active agents was reported for several systems26. A similar decrease for mixed systems was found with increase in alkyl chain length. The inclusion of two methylene groups seems to reduce the CMC by an approx. constant factor in both cases, due to the step-wise increase in hydrophobicity. The contribution of one CH2 group towards the free energy of micellization process of pure CnTABr has been discussed in detail26. The trend in aggregation number, Nagg of the micelles of cationic surfactants and their equimolar mixtures with NaC with alkyl chain length of the quaternary salts is shown in Fig. 3. While Nagg of cationic surfactants increases more steeply, only a moderate increase is found for the mixtures in line with Nagg for some such mixed micelles, estimated by light scattering3. The Nagg of mixed micelles is quite small (Nagg = 14-40), as compared to the respective pure cationic surfactants. The presence of NaC initially favours micellization of cationic surfactants, mainly due to electrostatic interaction. However, electrostatic interaction also offers constraints in the accommodation of several bile salt molecules into the micelles due to the bulkiness of the planar steroid non-polar part of bile salt. The Nagg of cationic micelles and mixed micelles along with some results on aggregation number in 1.0 M NaCl are shown in Table 2. The pyrene monomer fluorescence life-time (τ0) and first-order quenching rate constant (Kq) for CnTABr+NaC mixed systems in water as a function

Fig. 4—Pyrene fluorescence life-time (and quenching constant) vs. alkyl chain length plot for CnTABr+NaC; [(■) CnTABr; and (□) CnTABr +NaC] Table 2—Aggregation numbers, Nagg for micelles of cationic surfactants and their equimolar mixtures with sodium cholate (NaC) in water and 1.0 M NaCl at 30°C Surfactant C10TABr C12TABr C14TABr C16TABr C18TABr C10TABr + NaC C12TABr + NaC C14TABr + NaC C16TABr + NaC C18TABr + NaC

In Water (±2) 35 63 83 108 230 13 22 26 31 35

Nagg

In 1.0 M NaCl (±2) 73 90 143 27 26 33 38

of number of carbon atoms in the alkyl chain are shown in Fig. 4. No significant variation in τ0 is observed, which remains nearly constant (248±3 ns), showing that the probe senses a similar microenvironment in all cases. The substantial increase in life-time compared to the pure cationic micelles (118±3 ns) is mainly due to the shielding of excited state pyrene by the cholate anions from quenching species, such as O2 molecules and Br- ions, present in aerated solutions. In small spherical micelles Kq is reported to be roughly inversely proportional to the Nagg27. The variation of Kq in the present experiments suggests that size of host micelles affects the decay rate of quenching process and Kq decreases with increase in the alkyl chain length. The dye solubilization results for these mixed micelles in terms of absorbance of solubilized dye as a function of total surfactant concentration are shown

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111

in Fig. 5. Below CMC, the dye is not solubilized (in accordance with the CMCs of cationic surfactant+NaC observed from surface tension data), whereas above the CMC, a progressive linear increase in solubilization takes place. The solubilization is more for surfactants with larger alkyl chain length, which is quite expected23. However, compared to cationic surfactants alone, solubilization of the dye by mixed micelles was less due to their smaller size and less charge. This unusual behaviour results from the low value of Nagg in mixed micelles as discussed before. Mixed micelles The CMCs of cationic surfactants, NaC and their equimolar mixtures were used to calculate mole fraction of these two components in micelles and interaction parameter, β, which are often used in mixed surfactant systems. Interaction parameter β parameter was established to study the molecular interaction of the components of the mixed micelle. Higher the value of a β with negative sign, the larger is the molecular interaction between the components. According to Rubingh18, the mixed CMC (C12) for a binary surfactant system obtained by mixing two surfactants is given by the equation:

α (1 − α1 ) 1 = 1 + C12 f1C1 f 2 C2

... (2)

where α1 is the mole fraction of surfactant 1 in the total mixed solute; f1 and f2 are the activity coefficients and C1 and C2 are the CMCs of surfactants 1 and 2, respectively. As proposed by Clint28 for ideal mixed micelles, f1 = f2 = 1 and hence Eq. (2) reduces to the form: 1 α1 (1 − α1 ) = + C12 C1 C2

... (3)

Mixed CMC (C12) was calculated by using the Eq. (3) for ideal behaviour. By considering the phase separation model for micellization, Rubingh18 derived the relation:

(X1 ) 2 ln[(α1C12 /X1C1 )] =1 (1 − X1 ) 2 ln[(1 − α1 )C12 /(1 − X1 )C 2 ]

... (4)

where X1 is the mole fraction of surfactant 1 in the mixed micelle. Eq. (4) was solved iteratively to obtain

Fig. 5—Optical density vs. surfactant concentration plot for the solubilization of an azo Orange OT [(●) C10TABr + NaC; (*) C12TABr + NaC; (▲) C14TABr + NaC; (□) C16TABr + NaC; and (■) C18TABr + NaC] Table 3—CMCs, mole fraction of NaC in solution (∝), mole fraction in the mixed micelle (XNaC) and interaction parameter, β of equimolar mixtures of alkyltrimethylammonium bromide and NaC in water at 30°C Surfactant

C10TABr + NaC C12TABr + NaC C14TABr + NaC C16TABr + NaC C18TABr + NaC

CMC mM (± 0.05)

∝

XNaC

β

5.80 0.90 0.57 0.17 0.10

0.5 0.5 0.5 0.5 0.5

0.6 0.52 0.46 0.40 0.38

- 5.1 -10.2 - 9.08 -10.5 -11.5

the value of X1, from which β was evaluated, using the relationship: β=

ln[(α1C12 /X1C1 )] (1 − X1 ) 2

... (5)

The β values calculated for five mixed systems studied are listed in Table 3. These values range from -5 to -12, indicating strong attractive interaction (synergism). The value for C10TABr+NaC was less negative, showing weakest interaction. Moreover, the synergistic effects are observed to increase with increasing tail length of cationic surfactant. The calculated value of mole fraction of NaC in the micelles (XNaC) for equimolar mixture (XNaC = 0.5) for C10TABr is 0.6, indicating that the mixture contains NaC-rich micelles. For C12TABr-NaC (X=0.52) that means that composition of two surfactants in bulk water is the same as in the mixed micelles. For higher

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alkyltrimethylammonium bromides, mole fraction of NaC in mixed micelles is less than 0.5 and decreases with increasing carbon chain length in cationic surfactant. In these cases, the micelles are rich in cationic surfactants. These compositions of micelles can be correlated to the CMCs of cationic surfactants relative to NaC. Conclusion The role of bile salts as physiological surfactants is based on their micelle-forming properties. The mixed CMCs (cationic surfactants + NaC) are found to be far less than the individual surfactants, indicating the synergism. The interaction parameter β obtained from analysis of data using Rubingh’s theory showed strong interaction between NaC and cationic surfactants. Fluorescence-quenching results indicate the formation of small-sized mixed spherical micelle with aggregation number much less than micelles of cationic surfactant, which is well supported by dye solubilization results. Acknowledgement Financial assistance from CSIR Project No01/1827/02/EMR-II to one of the author PB is gratefully acknowledged. References 1 Small D M (1971) in The Bile Acids (Nair P P & Kritchevsky D, eds.), Vol 1, Plenum Press, New York 2 Gupta P M, Bahadur P & Singh S P (1979) Indian J Biochem Biophys 16, 36-37 3 Barry B W & Gray G M T (1975) J Colloid Interface Sci 52, 327-339 4 Ueno M, Kimoto Y, Ikeda Y, Momose H & Zana R (1987) J Colloid Interface Sci 117, 179-185 5 La Mesa C, Khan A, Fontell K & Lindman B (1985) J Colloid Interface Sci 103, 373-389

6 Vethamuthu M S, Almgren M, Brown W & Mukhtar E (1995) J Colloid Interface Sci 174, 461-479 7 Vethamuthu M S, Almgren M, Karlsson G & Bahadur P (1996) Langmuir 12, 2173-2185 8 Vethamuthu M S, Almgren M, Mukhtar E & Bahadur P (1992) Langmuir 8, 2396-2404 9 George A, Vora S, Desai H & Bahadur P (1998) J Surf Detg 1, 507-514 10 Scamehorn J F (1986) in Phenomena in Mixed Surfactant Systems (Scamehorn J F, ed), ACS Symposium Series 311, American Chemical Society, Washington, DC 11 Ogino K & Abe M (1992) in Mixed Surfactant Systems (Holland P M, Rubingh D N, eds.), ACS Symposium Series, American Chemical Society, Washington, DC 12 Holland P M & Rubingh D N (eds.) (1992) Mixed Surfactant Systems ACS Symposium Series, Vol. 501, American Chemical Society, Washington, DC 13 Yaacob I I & Bose A (1996) J Colloid Interface Sci 178, 638-647 14 Bhat M & Gaikar V G (1999) Langmuir 15, 4740-4751 15 Tomasic V, Stefanic I & Filipovic-Vincekovic N (1999) Colloid Polym Sci 277, 153-163 16 Bergstrom M (2001) Langmuir 17, 993-998 17 Gandhi H, Varade D & Bahadur P (2002) Tenside Surf Detg 39, 16-19 18 Rubingh D N (1979) in Solution Chemistry of Surfactants, (Mittal K L, ed), Vol. 1, pp. 337, Plenum Press, New York 19 Mukerjee P & Mysels K J (1971) Critical Micelle Concentrations of Aqueous Surfactant Systems, NSRDCNBS-36, Washington, DC 20 McGrath K M (1995) Langmuir 11, 1835-1839 21 Carnero Ruiz C & Aguiar J (2000) Langmuir 16, 7946-7953 22 Tanford C (1980) The Hydrophobic Effect-Formation of Micelles and Biological Membranes, 2nd edn., Wiley, New York 23 Jungerman E (1969) Cationic Surfactants, Marcel Dekker, New York 24 Bakshi M S (2000) Colloid Polym Sci 278, 1155-1163 25 Bakshi M S (2000) J Colloid Interface Sci 227, 78-83 26 Ruso J M & Sarmiento F (2000) Colloid Polym Sci 278, 800804 27 Zana R (1987) Surfactants in Solution; New Methods of Investigation, Marcel Dekker, New York 28 Clint J H, (1975) J Chem Soc Faraday Trans I, 71, 1327