5838
Langmuir 2003, 19, 5838-5845
Interface-Subphase Interactions of Rhamnolipids in Aqueous Rhamnose Solutions Su¨mer Peker,* S¸ erife Helvacı, and Gu¨nseli O ¨ zdemir Chemical Engineering Department, Ege University, Bornova, 35100 I˙ zmir, Turkey Received December 13, 2002. In Final Form: March 11, 2003
Effects of the presence of rhamnose (RH) sugar in the bulk phase on the interfacial properties of rhamnolipids (RL) are investigated in this work. Rhamnolipid compositions chosen to be studied were R1, R2, and their mixtures in the ratios of R2/R1 ) 1.2 and R2/R1 ) 1.07. Rhamnose in the bulk phase had a constant ratio (RH/RL) of 0.5, 1, 2, or 3 to the rhamnolipids. The results obtained from surface tension measurements were supported by surface shear viscosity and conductivity measurements. The interactions between rhamnolipids were confirmed with photographs taken under cross-polarized light. The overall effect of these properties was observed in the foam ability and stability of the solutions. The effect of rhamnose in the bulk phase is dependent on the composition of the monolayer; significant variations are observed with pure R1 and R2. In the rhamnolipid mixtures, rhamnose molecules penetrate into the monolayer at very low concentrations. At higher rhamnolipid concentrations, rhamnose molecules form bridges between the rhamnolipid molecules, lending rigidity to the monolayer.
Introduction Rhamnolipids produced by Pseudomonas aeruginosa are one of the most extensively investigated glycolipids. Research results on the efficiency of the rhamnolipids in the reduction of surface and interfacial tension,1 solubilization of oil,2 biological activity toward Gram positive and some Gram negative bacteria,3 zoosporic phytopathogens,4 and ability to form pH-sensitive vesicles5 open up many prospective application areas.6,7 The production methods, chemical structures, and surface activity of the isolated rhamnolipids are well documented.1,3,8 As the rhamnolipids are excreted directly into the nutrient medium, surface tension measurements can be used to monitor the progress of the production process.3,9,10 Surface and interfacial tensions were also measured in distilled and buffered saline solutions to assess the surface activity of the different types of rhamnolipids1 for use in oil recovery and soil remediation applications or to investigate the effects of the counterion.11 But as of yet no data have been reported in the literature on the effects of sugars, or in general polyalcohols, on the interfacial activity of rhamnolipids. Early work12 on the effects of polyalcohols on the surface activity of surfactants showed glycerol in the subphase to (1) Syldatk, C.; Lang, S.; Wagner, F.; Wray, V.; Witte, L. Z. Naturforsch. 1985, 40, 51-602. (2) Zhang, Y.; Miller, R. M. Appl. Environ. Microbiol. 1995, 61, 22472251. (3) Abalos, A.; Pinazo, A.; Infante, M. R.; Casals, M.; Garcia, F.; Manresa, A. Langmuir 2001, 17, 1367-1371. (4) Stanghellini, M. E.; Miller, R. M. Plant Dis. 1997, 81, 4-12. (5) Ishigami, Y.; Gama, Y.; Nagahora, H.; Yamaguchi, M.; Nakahara, H.; Kamata, T. Chem. Lett. 1987, 763-766. (6) Lang, S.; Fischer, L. Design and Selection of Performance Surfactants; Karsa, D. R., Ed.; CRC Press: Sheffield, 1999; pp 67, 85. (7) Lang, S.; Wullbrandt, D. Appl. Microbiol. Biotechnol. 1999, 51, 22-32. (8) Yamaguchi, M.; Sato, A.; Yukuyama, A. Chem. Ind. 1976, 4, 741742. (9) Mata-Sandoval, J. C.; Karns, J.; Torrents, A. J. Chromatogr., A 1999, 864, 211-220. (10) Patel, R. M.; Desai, A. J. J. Basic Microbiol. 1997, 4, 281-286. (11) Ishigami, Y.; Gama, Y.; Ishii, F.; Choi, Y. K. Langmuir 1993, 9, 1634-1636. (12) MacArthur, B. W.; Berg, J. C. J. Colloid Interface Sci. 1979, 68, 201-213.
expand the already expanded monolayers and condense compact monolayers. Dissolution of the surfactant during compression of the monolayer was also noticed.12 The interaction between the surfactant molecules in the monolayer with the polyalcohols dissolved in the bulk phase is an important issue from the standpoint of foambased food technology. Sugars in the subphase were found to decrease the foaming capacity of the surfactant but were found to increase the stability of the foam produced. To elucidate the mechanisms through which the foam stability was increased, the interactions of the glycerylmonostearate monolayer at the air-solution interface with the sugars, glucose, fructose, and sucrose in the bulk phase were investigated,13 as an example of a surfactant-sugar system commonly used in foods. The effects of sugars on the surface pressure-area isotherms of the surfactant obtained in a Langmuir balance were interpreted as an expansion of the monolayer at low surface pressures due to penetration of the sugars between the lipids and as barrier formation by the sugar molecules beneath the monolayer at higher surface pressures. Sugars were also found to affect the mechanical properties of the film by causing an increase in the coefficient of elasticity of the monolayer. Sugars and glycolipids play a vital role in cell functions,14 which roused interest on the interaction of sugar-based surfactants. Studies on gangliosides, glycolipids incorporating sialic acid groups, showed that the expanded monolayer becomes compressed when the charge on the sialic acid groups is neutralized at low pH values.15 A subphase molecule cannot penetrate a glycolipid monolayer if it is closely packed at the interface.16 An investigation of the stereochemistry of the hydrophilic groups of glycolipids17 has elucidated the role of the (13) Patino, J. M. R.; Dominguez, M. R.; de la Fuenta Feria, J. J. Colloid Interface Sci. 1993, 157, 343-354. (14) Murray, R. K.; Granner, D. K.; Mayes, P. A.; Rodwell, V. W. Harper’s Biochemistry, 32nd ed.; Appleton and Lange: Stamford, CT, 1990. (15) Luckham, P.; Wood, J.; Froggatt, S.; Swart, R. J. Colloid Interface Sci. 1993, 156, 164-172. (16) Heywang, C.; Mathe, G.; Hess, D.; Sackmann, E. Chem. Phys. Lipids 2001, 113, 41-53.
10.1021/la0269964 CCC: $25.00 © 2003 American Chemical Society Published on Web 06/11/2003
Rhamnolipids in Rhamnose Solutions
position of the hydroxyl groups on the sugar moieties: If the hydroxyl groups are laterally oriented, strong interactions are expected to be present among the surfactant molecules at the interface. Increase in the size of the sugar groups was found to favor the bilayer formation of glycolipids. In this work interactions of rhamnolipids in the monolayer at the air-aqueous solution interface with polyalcohols dissolved in the subphase are to be investigated. As an initial study, rhamnose was chosen as the polyalcohol due to the similarity in structure with the rhamnosyl groups of the surfactants. As no work on rhamnolipid-polyalcohol interactions is available in the literature, other physical properties of the solution, such as conductivity, viscosity, surface viscosity, and the rate of foam formation were determined either to elucidate or to confirm the results obtained through surface tension measurements. Materials and Methods
Langmuir, Vol. 19, No. 14, 2003 5839 developed by Wasan and co-workers.18 The viscometer was manufactured of stainless steel with a smooth surface finish. The inner and outer diameters of the cylindrical channel were 100 and 122 mm with a gap width of 10.94 mm. The same volume of solution was used in the experiments to keep the ratio of the width of the channel to the depth of the liquid nearly constant and greater than 2/π, the requirement for deep channels. The solutions were prepared in the same concentration range as in the surface tension measurements. All the rhamnolipid solutions were examined under polarized light with an Olympus BX 50 microscope. For this, thin films of solution were spread on glass slides and left to dry. Structure evolution during the drying period was observed under crosspolarized light and was recorded by photography. To see how the effect of rhamnose found on the interfacial properties would show up in applications, foaming properties of rhamnolipids were also determined. Foaming ability and stability of rhamnolipid solutions were determined by revising the method of Bikerman19 in a 32 mm diameter glass column with a height of 500 mm. Air was sent to the column through a 32 mm diameter sinter glass plate with 16 µm pore size, at the bottom of the column. Air flow was started before pouring the rhamnolipid solution (1 mL) from the top of the column to prevent early drainage. The foam volume and the flow rate of air through the column were recorded as a function of time. When the foam volume reached its maximum value, manifested by the constant height of the foam column and the bursting of air bubbles on the surface, air flow was stopped. Then the decay in the foam volume was measured as the variation of the height of the foam column with time.
Materials. The rhamnolipids used in this work were kindly donated by Jeneil Biosurfactant Co., Wisconsin, USA, of purity greater than 99%. The purity of the samples was further checked with thin-layer chromatography (TLC) analyses. The chemical structures of the rhamnolipids investigated are reported by Jeneil Biosurfactant Co., as 3-[3′-(L-rhamnopyranosyloxy)decanoyloxy]decanoic acid, R1, and 3-[3′-(2′′-O-R-L-rhamnopyranosyloxy)decanoyloxy]decanoic acid, R2. These molecules could also be denoted as R1C10C10 and R2C10C10, respectively, signifying the two decanoic acid groups making up the hydrophobic part of the molecules and one or two rhamnosyl groups in the hydrophilic part besides the carboxylic acid and carboxylate groups. The molecular weight of R1 is 504 g mol-1, and that of R2 is 650 g mol-1. The samples were sent in the form of solids (JBR 599) and as their 15% (wt) solutions in water (JBR 515). Pure R1 (lot no. 020130), R2 (lot no. 020401), and their mixtures in the molar ratio of 1.2 (lot no. 001010) and 1.07 (lot no. 010430) were used in this work. Rhamnose sugar was purchased from Merck in the form of L(+)-rhamnose monohydrate (RH) in a purity suitable for biochemical applications. Deionized water with specific electrical conductivity of κ ) 1 µS/cm was used throughout the experiments. The pH of the solutions prepared with this water had an average pH of 6.8. The viscosities were determined within (0.1 °C of the temperature desired for the specific case studied. All the other properties were determined at room temperature, 25 ( 2 °C. Methods. As rhamnolipids have short alkyl chains, surface tension measurements would give more reliable results than direct determination of the surface pressures in a Langmuir balance. Surface tensions were measured by the du Nou¨y ring method with a K86 Kru¨ss tensiometer. The accuracy of the tensiometer was controlled within (0.5 mN/m with the measurement of the surface tension of water at the start of each set of experiments. Preliminary experiments showed that the value of the surface tension reading remained constant after 30-40 min of equilibration time was allowed for the rhamnolipid molecules to adsorb on the interfaces. This equilibration time decreased with an increase in the concentration of rhamnolipid, reducing to 20 min at the critical micelle concentration (cmc). Two sets of solutions were prepared, and the surface tension measurements were made in parallel with two repetitions in each set to minimize experimental errors. The cmc values found by surface tension measurements were further checked by conductivity measurements. Specific conductivities κ, in µS/cm units, were measured with a WTW LF 330 digital conductometer (conductivity cell, TetraCon 325). Bulk viscosities were measured with a cross-arm Fuchs type of capillary viscometer. Surface shear viscosities were measured by the Deep-Channel Viscous Traction Shear Viscometer method
The aim of this work was to investigate the interactions of rhamnolipid molecules with each other and with rhamnose dissolved in the subphase through the hydroxyl groups. The hard sphere models and render forms of R1 and R2 molecules in their minimum energy positions obtained with ChemSite Pro software are given in parts a and b of Figure 1, respectively. These three-dimensional interactions were expected to affect the interfacial properties as well as some of the bulk properties. In this connection, the surface tensions of pure and mixed rhamnolipids were determined in the absence and presence of rhamnose in the subphase. Confirmation for the trends observed in surface tension profiles was sought through the determination of another surface property, surface shear viscosity, and the bulk properties of conductivity, bulk viscosity, and phase structure under cross-polarized light. Effect of Rhamnose on the Surface Tensions of Rhamnolipid Mixtures. Surface tension measurements give a good indication of the interactions between rhamnolipids in the monolayer and rhamnose dissolved in the bulk aqueous phase. To elucidate the interactions between rhamnose and rhamnolipids in this work, their molar ratio (RH/RL) was used as a parameter. The value of this ratio was held constant at 0.5, 1, 2, and 3 for all concentrations of rhamnolipids in a given set of experiments. The surface tensions of the pure components R1 and R2 are given in Figure 2a. The shape of the surface tension profiles is typical of anionic surfactants. R1 is consistently more surface active than R2 up to the critical micelle concentration, which has the same value of 1.5 × 10-4 mol/L for both R1 and R2. The minimum surface tensions at the cmc differ slightly with σcmc ) 29.7 mN/m for R1 and σcmc ) 32 mN/m for R2. Comparison of the values found in this work with the literature data showed the best agreement to be with the work of Abalos3 et al., who
(17) Hinz, H.; Kuttenreich, H.; Meyer, R.; Renner, M.; Fru¨nd, R.; Koynova, R.; Boyanov, A.; Tenchov, B. G. Biochemistry 1991, 30, 51255138.
(18) Wasan, D. T.; Gupta, L.; Vora, M. K. AIChE J. 1971, 17, 12871295. (19) Bikerman, J. J. Foams; Springer-Verlag: Berlin, 1973.
Results and Discussion
5840
Langmuir, Vol. 19, No. 14, 2003
Peker et al.
Figure 1. The hard sphere models and render forms of (a) R1 (R1C10C10) and (b) R2 (R2C10C10) molecules in their minimum energy positions.
found the cmc value of R2 as 1.63 × 10-4 mol/L and σcmc ) 28.8 mN/m. Variation of surface tensions of R1 and R2 with rhamnose in the subphase at a molar ratio of RH/RL ) 1/1 is also given in Figure 1a. The shape of the surface tension profile of RH/R1 ) 1 is significantly different from that of the pure component R1: After a gradual decrease in surface tension, a sharp decline is observed above a concentration of 3 × 10-6 mol/L ending up at a σcmc value of 28.6 mN/m, at cmc ) 0.7 × 10-4 mol/L. In the case of R2, surface tension at a molar ratio of RH/R2 ) 1 remains almost constant below 1 × 10-6 mol/L, after which it decreases with a greater slope than that of the pure component R2, reaching a minimum value of 34 mN/m at cmc ) 0.5 × 10-4 mol/L. Comparison of surface tension profiles with and without rhamnose displays different trends for R1 and R2. In the case of R2, the presence of rhamnose causes a decrease in surface tension throughout the concentration range up to the cmc value of 0.5 × 10-4 mol/L, which is less than half the value of the pure component. Despite the apparent augmentation in surface activity, the minimum surface tension is higher than that of pure R2. On the other hand, the presence of rhamnose causes the surface tension of pure R1 solutions to increase by about 10 mN/m. The two surface tension profiles intersect at a concentration of 1 × 10-5 mol/L due to their different rates of decrease in surface tension with rhamnolipid concentration. At higher concentrations, the presence of rhamnose causes a decrease in surface tension. It can be hypothesized that these trends arise from the different configurations of the rhamnose molecules with respect to the rhamnolipids in the monolayer. The two rhamnosyl groups of R2, folded under the molecule in Figure 1b, attract other rhamnose groups from the subphase, which bridge together the rhamnolipid molecules through hydrogen bonds. This stabilization results in a lowering of surface tension throughout the concentration range, up to the cmc. As these rhamnose bridges prevent the further compaction of the monolayer, the final surface tension reached is not as low as that of the pure R2 solution. The single rhamnosyl group of R1 cannot hold the rhamnose molecules in the subphase, which can come up to the monolayer, and penetrate between the rhamnolipid molecules. These intruding molecules pre-
Figure 2. (a) Effect of rhamnose on the surface tension of pure rhamnolipid solutions R1 and R2. (b) Surface tension of mixed rhamnolipid solutions with R2/R1 ) 1.07 as a function of the RH/RL ratio. (c) Surface tension of mixed rhamnolipid solutions with R2/R1 ) 1.2 as a function of the RH/RL ratio.
clude the compaction of the monolayer, causing an apparent increase in surface tension. At bulk concentrations greater than 1 × 10-5 mol/L, the array of rhamnolipids in the monolayer does not permit the penetration of rhamnose molecules, and this compaction in the monolayer causes a decrease in the surface tension. A similar model was proposed for other sugar-surfactant systems in the literature.13 The effects of rhamnose on the surface tension of rhamnolipids at a molar ratio of R2/R1 ) 1.07 and R2/R1 ) 1.2 are given in parts b and c of Figure 2, respectively. The surface tensions of rhamnolipid mixtures are similar in shape and fall between the values of the surface tension of the pure components of R1 and R2. At higher concentrations, the surface tension of the mixtures approach those of R2 solutions. In the absence of rhamnose, the minimum surface tension (29.7 mN/m) and the cmc (1.5 × 10-4 mol/L) remain the same as those of the pure components. The effect of the presence of rhamnose is more subtle due to the heterogeneity of the monolayer composition. The values of the cmc and the minimum surface tension at cmc are given for the pure components and their mixtures, at various RH/RL ratios in Table 1. Here the symbol (RL) denotes the rhamnolipids irrespective of their compositions. The cmc values are also given
Rhamnolipids in Rhamnose Solutions
Langmuir, Vol. 19, No. 14, 2003 5841
Table 1. The cmc Values and Minimum Surface Tensions Obtained for the Mixtures of R1 and R2 in the Presence of Rhamnose composition of the solution
cmc (mg/L)
cmc (mol/L) × 104
γcmc (mN/m)
RL ≡ R1 RH/RL ) 1 RL ≡ R2 RH/RL ) 1 RL ≡ (R2/R1 ) 1.07) RH/RL ) 0.5 RH/RL ) 1 RH/RL ) 3 RL ≡ (R2/R1 ) 1.2) RH/RL ) 0.5 RH/RL ) 1 RH/RL ) 2 RH/RL ) 3
75.6 35.3 87.5 40.9 115.9 58 58 115.9 116.7 116.7 116.7 116.7 116.7
1.5 0.7 1.5 0.5 1.5 1.0 1.0 2.0 1.5 2.0 2.0 2.0 2.0
29.7 28.6 32.0 34.0 29.7 31.5 31.2 28.0 29.7 30.0 29.7 29.6 33.3
Figure 3. Coefficient of elasticity of mixed rhamnolipid solutions with R2/R1 ) 1.07 as a function of the RH/RL ratio.
in (mg/L) units in the second column for ease in comparison with the literature values given in these units for the mixtures of rhamnolipids produced by Pseudomonas aeruginosa. Except for RH/RL ) 3, the extent of variation in surface tension is not significant and is independent of the R2/R1 ratio in the monolayer or the total concentration of the rhamnolipid. This behavior is preliminarily attributed to the bridging of rhamnolipid molecules by rhamnose through hydrogen bonds and formation of a barrier layer of rhamnose just beneath the monolayer. Surface tension of the solutions with rhamnose present in the ratio RH/RL ) 3 is sensitive to the composition of the monolayer: When R2/R1 ) 1.07, where the monolayer composition is approximately equimolar, surface tension decreases below that of the pure solution. The cmc also decreases to 1.0 × 10-4 mol/L. As the ratio of R2 molecules in the monolayer composition increases to R2/R1 ) 1.2, the surface tension with RH/RL ) 3 increases above the values obtained in the absence of rhamnose. A possible explanation could be the compaction effect brought about by the rhamnose molecules nested underneath the R1 molecules in the monolayer with an equimolar composition of R1 and R2 (R2/R1 ) 1.07) bridging them together through hydrogen bonds. Hard sphere model studies showed that in a staggered array of alternating R1 and R2 molecules, a hexagonal space becomes available where a rhamnose molecule could nest, playing a crucial role in interlinking the rhamnolipid molecules at the interface and rhamnose in the sublayer. On the other hand, the two rhamnosyl groups folded under the R2 molecules attract more rhamnose molecules from the bulk phase. The attractive forces exerted by the incoming rhamnose molecules lead to the dissolution of the R2 molecules from the monolayer, causing a decrease in the surface concentration and an increase in the surface tension. This explanation relies on the assumption that the monolayer composition is the same as the bulk phase composition and will remain speculative until the monolayer composition can be determined independently. To interpret the surface tension results quantitatively, the surface concentrations are calculated using the Gibbs adsorption equation
Γ)-
C ∂γ nRT ∂C
( )
T
(1)
from which the area occupied by a rhamnolipid molecule in the monolayer is found
A ) 1020/ΓΝ
(2)
Surface pressures, Π, are taken as the difference between the surface tension of pure water and that of the solution
Π ) γw - γ
(3)
The coefficient of elasticity, E, and the inverse of the compressibility coefficient, κ, are used to quantify the compressibility of the monolayers under equilibrium conditions
E)
dΠ 1 ) -A κ dA
(4)
In these equations the units are as follows: surface tension γ, N/m; surface concentration Γ, mol/m2; universal gas constant R, J/(mol K); molecular area A, Å2/molecule; the Avogadro number, N ) 6.02 × 1023 molecules/mol; surface pressure Π, N/m; and elasticity coefficient E, N/m. At pH ) 6.8, the rhamnolipid molecules are almost completely dissociated, so n ) 2 in eq 1. Curve Expert software was used for the regressions required to evaluate the derivatives in eqs 1 and 4. The regressions were made within the range of concentrations below the cmc, where maximum variations are observed. A provision has to be made at this point: Gibbs adsorption isotherm given by eq 1 should be used when the surface tension changes as a function of the concentration of the surface active agent(s) only. It is inadequate in taking into account the effect of another solute in the bulk phase or in the sublayer, for which another term is necessary in the equation of the isotherm. Surface equations of state developed by Lim and Berg20 take into account the effect of solutes in the bulk phase, but in a later paper,12 the equations were found not to correlate well for polyalcohols such as D-glucose, which were solid at the temperature of the measurement. Since rhamnose is also a solid at room temperature, the equations could not be used. The coefficients of elasticity of the pure components R1 and R2 and their mixtures are given in Figure 3 to illustrate the variation with rhamnolipid concentration. The limiting coefficients of elasticity at the cmc are summarized in Table 2. The value of the coefficient of elasticity of monostearin when water is the subphase13 is given as 145 mN/m at 25 °C and Π ) 40 mN/m. This value falls within the range given for the pure rhamnolipids and their mixtures in Table 2. Elasticity values at Π ) 10 and 25 mN/m were also found to be consistent with the present results. The agreement between these results is significant in terms (20) Lim, Y. C.; Berg, J. C. J. Colloid Interface Sci. 1975, 51, 162175.
5842
Langmuir, Vol. 19, No. 14, 2003
Peker et al.
Table 2. Limiting Coefficients of Monolayer Elasticity of the Pure Components R1 and R2 and Their Mixtures composition of the rhamnolipid monolayer
coefficient of elasticity (mN/m)
R1 R2 R2/R1 ) 1.07 R2/R1 ) 1.2
217 205 277 138
of the reliability of the surface tension measurements, as the coefficients of elasticity of monostearin were calculated based on measurements with a Langmuir type film balance, and those of rhamnolipids, based on surface tension measurements. The values in Table 2 show that except for R2/R1 ) 1.2, the rhamnolipid monolayers are more rigid than the monostearin layers. This result is to be expected from the stereochemistry of rhamnolipids, which have many hydroxyl groups in their hydrophilic parts oriented in different directions. Effect of Rhamnose on the Surface Shear Viscosities of Rhamnolipid Mixtures. Surface shear viscosity is another physical property that gives an indication of the resistance of the monolayer toward deformation under the action of applied shear. The measurement technique is based on determining the relative velocities of the base plate and a minute Teflon particle floating at the surface.18,21 Strict quantitative comparisons cannot be made with this technique, but qualitatively the trends in the surface shear viscosity variations correlate inversely with the trends in the variation of surface tension values. The variation of the surface shear viscosity of mixed monolayers as a function of the RH/RL ratio and rhamnolipid concentration is given in parts a and b of Figure 4 for R2/R1 ) 1.07 and 1.2, respectively. The values of the surface shear viscosities are in the same range of an aqueous solution of 0.01% sodium lauryl sulfate mixed with lauryl alcohol.21 The decrease in surface viscosity around cmc may be due to compaction of the monolayer: The hydrocarbon chains of the rhamnolipids in close proximity, due to hydrogen bonds between the hydrophilic groups, may seem equivalent to a hydrocarbon surface to the Teflon particle which floats on the surface. Since the surface shear viscosity of all pure liquids is accepted to be zero, the decrease in the values around the cmc concentration seems to indicate such a compaction. At concentrations below the cmc, when the monolayer is not fully packed so as to exhibit organic phase characteristics, a decrease in surface tension causes a concomitant increase in the monolayer surface shear viscosity. Indeed, both properties are different manifestations of the degree of compaction of the monolayer: In the absence of rhamnose, a greater decrease of surface shear viscosity with increasing rhamnolipid concentration in the case of R2/R1 ) 1.07 in comparison with R2/R1 ) 1.2, signifies a greater compaction in the former case. The values of the coefficient of elasticity E, in Figure 3 corroborate the trends in the surface shear viscosity. In all the cases presented in Figure 4, the maximum effect of the presence of rhamnose is observed at the ratios of RH/RL ) 1 and 2. Different trends in surface shear viscosity are observed at a ratio of RH/RL ) 3 with values less than that of the pure solution in the case of R2/R1 ) 1.2, and greater, in R2/R1 ) 1.07, correlating inversely with the surface tension measurements. Effect of Rhamnose on the Bulk Phase Properties of Rhamnolipid Mixtures. The bulk phase properties, (21) Mohan, V.; Gupta, L.; Wasan, D. T. J. Colloid Interface Sci. 1976, 57, 496-504.
Figure 4. (a) Effect of rhamnose on the surface shear viscosity of mixed rhamnolipid solutions with R2/R1 ) 1.07. (b) Effect of rhamnose on the surface shear viscosity of mixed rhamnolipid solutions with R2/R1 ) 1.2.
Figure 5. Effect of rhamnose on the conductivities of pure and mixed rhamnolipid solutions.
namely, conductivity and viscosity of the solutions and the texture observed under cross-polarized light, were determined to supplement the interactions found in the monolayer. Conductivity measurements are used to determine the binding energies and critical aggregation concentrations in the literature.20-24 The variation in conductivity of the rhamnolipid solutions is given in Figure 5. Conductivity of the solutions is proportional with the number of charged colloidal particles within the solution. Thus, the beginning of a steep rise in conductivities signifies the formation of aggregates in the bulk phase. In the absence of rhamnose, the conductivities of the solutions start to increase below the cmc concentration and increase smoothly at a greater rate as the cmc value is approached. This gradual, smooth increase is also observed in the case of rhamnolipid (22) Caboi, F.; Chittofrati, A.; Lazzari, P.; Monduzzi, M. Colloids Surf., A 1999, 160, 47-56. (23) Minatti, E.; Zanette, D. Colloids Surf., A 1996, 113, 237-246. (24) Bakshi, M. S.; Kaur, G. J. Surfactants Deterg. 2000, 3 (2), 159166.
Rhamnolipids in Rhamnose Solutions
Langmuir, Vol. 19, No. 14, 2003 5843
Figure 6. Photographs taken under cross-polarized light.
mixtures with RH/RL ) 3. However, when rhamnose is present at a ratio of RH/RL ) 1, an initial increase of conductivity is observed at 1 × 10-5 mol/L concentration, a gradual approach to an equilibrium value and then a very sharp increase at the cmc concentration of 5 × 10-5 mol/L. These observations are in parallel with the surface tension results presented in Figure 2a, where the surface tension profile of RH/R1 ) 1 intersects the pure component surface tension profile and then continues to decrease sharply down to the cmc concentration. The agreement between these observations implies the initiation of aggregate formation and premicellization in the bulk phase before micelle formation takes place.25 Viscosity measurements showed that the viscosities of the solutions do not increase beyond 7 cP, even at the solubility limit of RL. The solutions exhibited Newtonian rheology. The differences in the viscosities of the solutions in this range of concentrations were not large enough to discriminate the effect of the composition of the rhamnolipids (R2/R1 ratios) or the sugar content (RH/RL ratios). The appearance of rhamnolipid mixtures under crosspolarized light is given in Figure 6. Investigation of the solutions of R1 and R2 under cross-polarized light showed the existence of typical maltese crosses designating a lamellar nematic (NL) phase.22,26 The schlieren texture of the mixed rhamnolipids R2/R1 ) 1.07 in Figure 6c is indicative of a nematic calamitic (NC) phase, whereas the structure observed in R2/R1 ) 1.2 (Figure 6d) indicates (25) Exerowa, D.; Nikolov, A. Surfactants in Solution; Plenum Press: New York, 1984; Vol. 2, p 1313. (26) Kuzma, M. R.; Saupe, A. Structure and Phase Transitions of Amphiphilic Lyotropic Liquid Crystals. In Handbook of Liquid Crystals Research; Collings, P., Patel, J. S., Eds.; Oxford University Press: Oxford, 1997; Chapter 7.
lamellar D phase. Presence of rhamnose caused all the solutions to appear isotropic under cross-polarized light, as can be observed in the photomicrograph in Figure 6e. All of the textures are closely related and confirm the interactions existing between the rhamnolipid molecules and the effect of rhamnose. Effect of Rhamnose on the Foaming Behavior of Rhamnolipid Mixtures. Foaming behavior is taken up in this work to see the cooperative effect of the surface and bulk phase properties determined separately. There are many methods developed for determining the different aspects of the foaming ability and stability of foams in the literature.27,28 Of these, the standardized Ross-Miles test is used to characterize stable foams, through measurement of foam volume immediately after formation, and foam stability, by measurement of the height of foam as a function of time. The method of Bikerman is used to characterize transient foams through the average lifetime of a bubble which is the slope of the linear curve obtained by plotting the volume of foam formed as a function of the volumetric flow rate of air. In this work, rhamnolipid foams could be obtained only within a very narrow range of air flow rates: Due to molecular interactions between the molecules, foam did not form below this range. Above the upper limit of air flow, an expanded polyhedral foam formed on the sintered glass plate at the entrance of the glass column, on which an unexpanded spherical foam bubble column nested. In time, the expanded region collapsed. Within these limiting (27) Jo¨nsson, B.; Lindman, B.; Holmberg, K.; Kronberg, B. Surfactants and Polymers in Aqueous Solution; John Wiley and Sons: Chichester, 1999; Chapters 3 and 15. (28) Exerowa, D.; Kruglyakov, P. M. Foam and Foam Films; Elsevier Science: Amsterdam, 1998.
5844
Langmuir, Vol. 19, No. 14, 2003
Peker et al.
Figure 7. (a) Effect of rhamnose on the foaming ability and stability of rhamnolipid solutions with R2/R1 ) 1.07. (b) Effect of rhamnose on the foaming ability and stability of rhamnolipid mixtures R2/R1 ) 1.2. (c) Effect of rhamnolipid concentration on the formation rate of foams in mixed rhamnolipid solutions with R2/R1 ) 1.07. (d) Effect of rhamnolipid concentration on the rate of decay of foams in mixed rhamnolipid solutions with R2/R1 ) 1.07.
air flows, regularly expanding foam columns were obtained. Under these conditions, revision of the Bikerman method seemed the most appropriate way and was used in this work to bring forth the effect of the molecular interactions among rhamnolipids and rhamnose molecules. In the experiments, the foam was formed as in the original procedure and the variation in height were recorded as a function of time. When the height of the foam column reached a steady level, foam cells were observed to collapse at the upper surface of the foam column. At this point the air flow was stopped and the decrease in the height of the column was recorded as a function of time. This method of determining the foaming ability is not a standard method and in addition has the disadvantage that the drainage effect cannot be completely eliminated during the formation of foam. Nevertheless, excellent correlations could be obtained with the other surface properties. Examples for the complete range of variations in the foam volume during the experiments conducted under the same conditions of temperature (27 °C) and concentration (0.22 mol/L) are given in parts a and b of Figure 7 for pure R2/R1 ) 1.07 and 1.2, respectively, together with the effect of rhamnose at a molar ratio of RH/RL ) 0.5. Air flow rates are also given on the same figure as 10 times the actual value. The important features in this plot are (1) the rate of formation of the foam column given by the slope of the rising section, (2) the period over which the foam column retains its original volume, expressed by the duration of the plateau right after stopping the air flow, and (3) the mode of variation of the column height during the collapse of the column. These aspects of foam formation and stability are evaluated below: (1) Rate of Formation of the Foam Column. A necessary condition for foam formation is the elasticity of the foam film: the more elastic the film, the easier the
Table 3. Foaming Ability and Rates of Foam Formation of Pure Rhamnolipids and Their Mixtures rhamnolipid composition of the solution
max foam volume (cm3)
rate of foam formation (cm3/min)
R1 R2 R2/R1 ) 1.07 R2/R1 ) 1.2
241 233 129 145
2.61 3.44 1.86 2.98
foam formation.27 The rate of foam formation given by the initial slopes of the curves in Figure 7 correlate inversely with the coefficients of elasticity given in Table 1. As an example, the rates of foam formation of R2/R1 ) 1.07 (1.86 cm3/min) and of R2/R1 ) 1.2 (2.98 cm3/min) correlate inversely with the coefficient of elasticities 277 and 138, respectively, given in Table 1. The maximum volume of the foam and rates of foam formation are summarized in Table 3. It should be noted that the coefficient of elasticity in Table 1 is for the monolayer and not the film. These two are equivalent only in the case of Newton black films.28 Monolayer elasticity plays a significant role but is not the only parameter: film elasticity and dilational and shear viscosities of the monolayer are also effective in foam formation. Though of minor significance, viscosity of the solution unavoidably becomes a factor (µ1.2 > µ1.07) in determining the rate of foam buildup by reducing the drainage rate in the method chosen in this work. Other parameters that affect the rate of foam formation are concentration of the rhamnolipids and the RH/RL ratio, as illustrated in Figure 7c for the case of R2/R1 ) 1.07. The rate of foam formation increases with the concentration of the rhamnolipids. As both of the concentrations 0.01 and 0.2 M are much higher than the cmc, compaction of the film may not be the single operating factor. Availability and rate of diffusion of RL molecules to the
Rhamnolipids in Rhamnose Solutions
sight of the developing interface may also be a determining factor. Rhamnose decreases the formation rate under all conditions, especially when present in a ratio of RH/RL ) 3. At lower RH/RL ratios, the effectiveness in decreasing the rate of foam formation depends on the concentration: The effect of rhamnose decreases in compact films where the interactions between the rhamnolipid molecules are greater than those with rhamnose. (2) Period of Stability of the Foam Column. The maximum volume of foam formed gives the air entrainment capacity (cm3 of air/1 cm3 of solution) under the given conditions. Many factors affect the stability of the foam among which can be cited the bulk and surface viscosities and long range interactive (repulsion) forces. The first two parameters decrease the rate of drainage, and the third prevents further thinning. Cohesive forces in compact films lend stability to the foams. But the same forces enhance the growth of holes randomly formed within the thin film leading to the rapid collapse of the foam column.27,28 We see the operation of these interactive forces in the case of R2/R1 ) 1.07 and 1.2, where the periods of stability represent these two extreme cases of 80 min (time interval between 80 and 160 min) and zero (immediate initiation of collapse), respectively. Presence of rhamnose (RH/RL ) 0.5) moderates the stability period to 65 and 50 min, respectively, for the foams of R2/R1 ) 1.07 and 1.2, under consideration in Figure 7a,b. Interlinking of
Langmuir, Vol. 19, No. 14, 2003 5845
RL molecules through rhamnose intermediaries by hydroxyl bonds is probably the main cause for this stability, as an outcome of which more “uniform” periods of stability are obtained irrespective of the R2/R1 ratio. (3) Collapse of the Foam Column. The mode of the foam collapse depends on the concentration of the surfactant, as can be observed in Figure 7d. Under the action of interactive forces described above, the foam column collapses as an “avalanche” after a period of stability28 in highly concentrated systems. The linear decay of the foam in the case of low surfactant concentrations shows that drainage of the solution, which depends on the bulk and surface viscosities, and resistance of the liquid film toward gas diffusion29 control the rate of collapse of the foam column. Presence of rhamnose detains the decay of the foam at high rhamnolipid concentrations and speeds up the decay at low rhamnolipid concentrations. Acknowledgment. The authors are grateful for the support given to this work by the National Research Council of Turkey (TU ¨ BI˙ TAK), through Project MI˙ SAG 165, and to the Center for Scientific and Technological Research of Ege University (EBI˙ LTEM) through Project 2000/BI˙ L/020. LA0269964 (29) Yapar, S.; Peker, S. Colloids Surf. 1999, 149, 307-314.
