A PM-IRRAS Investigation of Monorhamnolipid Orientation at the Air

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A PM-IRRAS Investigation of Monorhamnolipid Orientation at the Air−Water Interface Hui Wang,† Clifford S. Coss, Anoma Mudalige, Robin L. Polt, and Jeanne E. Pemberton* Department of Chemistry and Biochemistry, University of Arizona, 1306 E. University Boulevard, Tucson, Arizona 85721, United States S Supporting Information *

ABSTRACT: The rhamnolipid biosurfactants have been considered as possible “green” alternatives to synthetic surfactants due to their greater compatibility with the environment and excellent surface active properties. In order to understand the molecular orientation of rhamnolipids at the air−water interface, a new monorhamnolipid with two octadecyl chains, Rha−C18−C18, has been studied at the air−water interface with polarization modulated-infrared reflection absorption spectroscopy (PMIRRAS). Since rhamnolipids possess a carboxylic acid, and hence exhibit pH-dependent properties, their water surface orientation is studied in solutions of pH 2, 5, and 8. Rhamnolipids have also been reported to form strong complexes with Pb2+; thus, the effect of the presence of Pb2+ on molecular orientation at the interface is also investigated. PM-IRRA spectra indicate an increase in alkyl chain order and a decrease in alkyl chain tilt angle as the surface pressure of the monolayer increases, with pHindependent tilt angles ranging from 63° to 45°. Molecular modeling using Spartan provides insight into the cause of this large tilt angle as being due to the nature of the monorhamnolipid packing at the air−water interface as dictated by its large hydrophilic headgroup.

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INTRODUCTION Rhamnolipids are surfactants first discovered in metabolites of Pseudomonas aeruginosa bacterial strains.1 They are composed of one or two rhamnose sugar moieties and a βhydroxyalkanoate alkanoic acid. Rhamnolipids exhibit less toxicity and higher biodegradability with comparable or better surface activity than traditional synthetic surfactants.2−6 As surface active agents, rhamnolipids reduce the surface tension of pure water from 72 to 25 mN/m,7,8 a performance that is better than that of most synthetic surfactants.9 Rhamnolipids are also very efficient at forming micelles in solution. Depending on the chemical composition of the rhamnolipid mixture under consideration, the critical micelle concentration (cmc) values for mixtures of rhamnolipids have been reported to vary from 50 to 200 mg/L.8,10−12 Dirhamnolipids possessing two rhamnose moieties tend to have smaller cmc values than the corresponding monorhamnolipids;8 rhamnolipids possessing sites of chain unsaturation have greater cmc values than the corresponding saturated surfactants,11 and rhamnolipids with longer fatty acid chains have smaller cmc values than those with shorter chains.10,12 Despite widespread interest in this class of biosurfactants, little work has been done to characterize highly purified rhamnolipids to illuminate their fundamental interfacial characteristics. In the work reported here, polarization modulation-infrared reflection absorption spectroscopy (PMIRRAS) is used to study the molecular orientation of adsorbed © 2013 American Chemical Society

monorhamnolipids (mRLs) at the air−water interface. Rhamnolipids (RLs), as surface active molecules, adsorb at the air−water interface in an oriented fashion. Surface tension measurements provide some insight into mRL adsorption behavior, including the adsorption constant and the molecular cross-sectional area. However, despite extensive study of these species, a dearth of molecular-level details about mRL adsorption persists. Of specific interest in this work is development of a molecular-level description of mRL orientation at the air−water interface. The molecular orientation of surfactants at interfaces plays a fundamental role in their surfactant properties in a wide range of technological applications.13 Therefore, orientation of mRLs at the molecular level is of importance in understanding their excellent performance in lowering surface tension and in possibly expanding application of these unique materials for a broader array of uses.

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EXPERIMENTAL SECTION

Materials. The mRL Rha−C18−C18 whose molecular structure is shown in Figure 1a was synthesized and purified using the procedure described in the Supporting Information. NMR and mass spectral data for the final Rha−C18−C18 product are also included in the Supporting Information (see Figures S1−S3). Rh−C18−C18 was Received: December 31, 2012 Revised: February 11, 2013 Published: February 13, 2013 4441

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Figure 1. (a) Molecular structure of Rha−C18−C18. (b) Schematic illustration of reflection at a three-layer system in which layer 1 is air, layer 2 is the surfactant thin film, and layer 3 is the subphase (water). The red arrows represent IR rays. Parallel (p) and perpendicular (s) polarized light are shown for incident radiation. 3.43 μm, J0(ϕ0) equals zero and at wavelengths (frequencies) nearby, values of J0(ϕ0) stay very small. For the specific peak frequencies of interest in this work of 2850 and 2920 cm−1 for the νs(CH2) and νas(CH2) bands, respectively, J0(ϕ0) values are on the order of 7 × 10−4 and zero, respectively (see Figure S4, Supporting Information), thereby rendering the second term in the denominator negligible compared to the first. With this simplification, the spectral data can be plotted as

dissolved in hexane (Sigma, Spectra Pure) to make a stock solution of 1 mg/mL for deposition of monolayers at the air−water interface. FTIR KBr Transmission Measurements. Rha−C18−C18 (1.3 mg) was mixed with 130 mg of dry IR-grade KBr and ground in an agate mortar, and 100 mg of this mixture was pressed into a pellet. The pellet thickness was measured by a micrometer to be an average thickness of 203 μm. PM-IRRAS Measurements. The PM-IRRAS experiments were performed using a Nexus 670 IR spectrometer (Thermo Scientific Corp.) equipped with an external tabletop optical module, MCT-A detector, photoelastic modulator (Hinds Instruments PM-90 with a II/ ZS 50 ZnSe 50 kHz optical head), and a synchronous sampling demodulator (GWC instruments).14 The polarization of incident infrared light is modulated at 100 kHz at 2918 cm−1, where the v(C− H) vibrational bands of interest are located. The best signal-to-noise ratio was achieved using an incident angle of 74°. Spectra were collected with a moving mirror velocity of 1.2 cm/s by coadding 10 000 scans at a resolution of 4 cm−1. Quantitative tilt and twist angle values reported here represent the average of four independent measurements. A Teflon Langmuir trough (Nima Technology) was used to control and maintain surface pressure during spectral acquisition. Rha−C18− C18 monolayers were formed by depositing a small volume (50 μL) of the stock solution in hexane (1 mg/mL) onto the water surface and allowing the hexane to evaporate for 15 min prior to compression. Additional stock solution was added as needed in some cases to reach the target pressure. Subphase pH was adjusted with HCl for acidic conditions and NaOH for basic conditions. Temperature was maintained at 20 °C throughout these experiments. PM-IRRAS spectra were processed in Grams 32 for baseline correction and peak-fitting. After Fourier transformation and ratioing, the differential reflectance spectrum obtained is given by14

S=

S(d) − S(0) ΔS = S S(0)

where S(d) and S(0) are the PM-IRRAS signals of the film-covered and film-free surface, respectively. Using this normalized difference quantity eliminates the second-order Bessel function, J2(ϕ0). Molecular Modeling. Molecular modeling was performed in Spartan 08 Version 1.2.0 (Wave function, Inc.) Molecular structures were energy-minimized using the molecular mechanics model available in the package.

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ELECTROMAGNETIC THEORY FOR INTERPRETATION OF PM-IRRAS DATA Due to the significant differences in the optical properties of water and metal surfaces, PM-IRRAS at the air−water interface is more complicated that at the air−metal surface. The PMIRRAS response represents a unique surface selection rule at the air−water interface that results in spectral bands that can occur in both positive-going and negative-going directions, as first summarized by Blaudez et al.17 Briefly, a positive-going band indicates a transition dipole moment (TDM) oriented parallel to the surface; a negative-going band indicates a transition dipole moment oriented along the surface normal; bands with intermediate tilt angles of the transition dipole moment range in amplitude from small to vanishing. As a result of this surface selection rule behavior, the strategy for quantitative determination of the average tilt angle for a collection of transition dipole moments at an interface using a given vibrational band is to identify the best match between the experimental differential reflection spectrum and a differential reflection spectrum simulated from first principles for different tilt angles.18,19 In this work, simulated spectra were calculated using a Mathcad program written in-house based on classical electromagnetic theory for an N-phase system of parallel, optically anisotropic layers.20,21 This theory is introduced briefly here. For the three-layer optical configuration shown in Figure 1b in which phase 1 is the ambient, phase 2 is the adsorbed

J2 (ϕ0)(R p − R s) (R p + R s) ± (R p − R s)J0 (ϕ0)

(2)

(1)

where J0(ϕ0) and J2(ϕ0) are zero- and second-order Bessel functions, respectively. The differential reflectance S is in the same form as obtained from demodulation with a lock-in amplifier.15,16 Although for PM-IRRAS at metal surfaces the magnitude of Rp − Rs is very small, rendering the second term in the denominator negligible compared to Rp + Rs, this is not true for PM-IRRAS on dielectric surfaces such as water, for which Rp − Rs is typically large. Therefore, proper consideration must be given to dealing with this term in such experiments. The most convenient approach to eliminating this term is to conduct the experiment under conditions at which J0(ϕ0) is very small. This can be accomplished by setting the phase shift ϕ0 = π at a wavelength close to the vibrational bands of interest. Here, this wavelength was chosen to be 3.43 μm (2918 cm−1). Under these conditions, as shown by Figure S4 in the Supporting Information, at 4442

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surfactant layer, and phase 3 is the water, the characteristic matrix for the second layer, M2, is expressed as follows: ⎡ −i sin β2l ⎤ ⎥ ⎢ cos β2l g 2l ⎥ M 2l = ⎢ ⎥ ⎢ ⎢⎣−ig2l sin β2l cos β2l ⎥⎦

(3)

where l indicates the polarization, s or p, β2l is the optical path difference in the second layer, and g2l is the polarizationdependent effective complex refractive index; β2l and g2l are defined as follows: β2s = 2πνd 2n2̃ y cos ϕ2s̃

(4)

g2s = n2̃ y cos ϕ2s̃

(5)

̃ β2p = 2πνd 2n2̃ x cos ϕ2p

(6)

g2p =

̃ cos ϕ2p n2̃ x

(7)

where d2 is the thickness of the second layer, and ϕ2,s and ϕ2,p are defined for s- and p-polarized light, respectively, by n1 sin ϕ1 = n2̃ y sin ϕ2s̃

(8)

̃ n1 sin ϕ1 = n2̃ z sin ϕ2p

(9)

The characteristic matrix for a three-layer system is defined as follows. ⎡ m11 m12 ⎤ M = M2 = ⎢ ⎥ ⎣ m21 m22 ⎦

Figure 2. (a) IR transmission spectrum of a 1 wt % KBr pellet of Rha− C18−C18. (b) Extinction coefficient, k, spectrum of Rha−C18−C18 obtained from IR transmission spectrum. (c) Refractive index, n, spectrum of Rha−C18−C18 obtained by Kramers−Kronig transformation of the spectrum in part b.

(10)

Using the components of M, the complex reflectivity, rl, can be expressed as rl =

{m11 + m12g31}g11 − {m21 + m22g31} {m11 + m12g31}g11 − {m21 + m22g31}

refractive index, although values could be different along different axes for the uniaxial film, nx, ny, and nz are generally taken to be equal to the isotropic refractive index, niso, because of the insensitivity of reflection−absorption values to the refractive index.19 Thus, since the directional optical constants of the anisotropic uniaxial Rha−C18−C18 thin film are known from the transmission experiment, the only parameters not known for the simulation of reflectance spectra are the tilt angle and the thickness of the thin film, d, which is a direct function of the tilt angle. To proceed with the simulation, therefore, the film thickness is estimated using lengths obtained from the energy-minimized molecular mechanics structure of Rha− C18−C18. From this structure, the length of the molecule is determined to be 2.5 nm. Thus, the film thickness, d, is estimated here to be the molecular length corrected for the tilt angle by d = (2.5 nm) cos(θtilt). With these parameters in hand, PM-IRRA spectra as a function of tilt angle can be predicted using this modified classical electromagnetic theory. A series of these predicted spectra for the ν(CH2) modes [νas(CH2) and νs(CH2)] with TDMs titled at angles ranging from 0° to 85° with respect to the surface normal are shown in Figure 3b. The simulated spectrum decreases in intensity as the tilt angle increases as the result of both changes in orientation and a decrease in monolayer thickness.

(11)

Reflectivity values observed in an optical measurement are represented by

R l = |rl|2

(12)

As a result, the reflection spectrum for a multilayer can be simulated if the complex anisotropic refractive indices of each layer are known. The relevant isotropic optical constants n and k for water are known and those for Rha−C18−C18 were determined in a transmission FTIR experiment on a KBr pellet of the Rha− C18−C18 powder according to established procedures22,23 using a Kramers−Kronig transformation as described in the Supporting Information.24 This spectrum is shown in Figure 2. A monolayer film formed at the air−water interface is generally structured with the molecules in a uniaxial configuration for which kx = ky. Thus kx = k y =

3 k iso sin 2 θ 2

kz = 3k iso cos2 θ

(13) (14)

where θ is the angle between a selected transition dipole moment and the surface normal (see Figure 3a). In terms of the 4443

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Figure 3. (a) Tilt angle θtilt defined as the angle between the hydrocarbon chain axis and the surface normal; θas and θs defined as the angles between the surface normal and the transition dipole moments (TDMs) of the νas(CH2) and νs(CH2) modes. (b) Simulated PM-IRRA spectra for a monolayer of Rha−C18−C18 oriented with TDMs of the νas(CH2) and νs(CH2) modes tilted at various θtilt ranging from 5° to 90° with respect to the surface normal.

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RESULTS AND DISCUSSION The surface pressure−area isotherm for Rha−C18−C18 on a water subphase of pH 2 in a Langmuir trough is shown in Figure 4. Three distinct regions of monolayer organization are

change in surface pressure. This isotherm shows that the monolayer reaches the solid-state phase at a surface pressure of ∼10 mN/m, above which the molecules are tightly packed in an ordered arrangement. To study further the surface-pressure dependence of molecular orientation within the Rha−C18−C18 monolayer, PM-IRRA spectra were collected at surface pressures of 10, 20, 30, and 40 mN/m. As noted above, Rha−C18−C18 has a carboxylic acid moiety, rendering its adsorption behavior at the air−water interface pH-dependent. Therefore, PM-IRRAS studies of the Rha−C18−C18 monolayer were performed on water subphases at pH of 2, 5, and 8. These pH values represent conditions below, at and above the Rha−C18−C18 pKa of ∼5.5.25 Since rhamnolipids are also known to be strong complexants of heavy metal cations,4 the effect of Pb2+ in the subphase on the molecular orientation of the Rha−C18−C18 monolayer was also investigated. A typical PM-IRRA spectrum from a Rha−C18−C18 monolayer together with its KBr transmission spectrum is shown in Figure 5. The broad negative-going bands at 1660 and 3560 cm−1 are due to the interfacial water layer.26 When the Rha−C18−C18 is deposited onto the water surface, these

Figure 4. Surface pressure−area isotherm for a Rha−C18−C18 monolayer on a water subphase at pH 2. Three regions of monolayer organization are indicated: gaseous, liquid, and solid phases.

immediately apparent upon examining the isotherm; these are assigned to Rha−C18−C18 in the gaseous, liquid, and solid states. In the gaseous state, Rha−C18−C18 molecules are randomly oriented at the interface and are widely spaced such that little intermolecular interaction is possible. As a result, upon compression of the barriers leading to a decreasing in area per molecule, little change in the surface pressure is observed. In the liquid state, the Rha−C18−C18 molecules are closer to each other, allowing intermolecular interactions. In the solid state, the Rha−C18−C18 molecules are close-packed and experience their maximum intermolecular interaction. At this stage, a small amount of compression causes a significant

Figure 5. (a) Transmission spectrum of a 1 wt % KBr pellet of Rha− C18−C18 with the major vibrational modes assigned. (b) Full PMIRRA spectrum of a Rha−C18−C18 monolayer on a pH 2 water subphase surface at a surface pressure of 40 mN/m. 4444

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Figure 6. PM-IRRA spectra from a Rha−C18−C18 monolayer at (a) pH 2, (c) pH 5, (e) 8, and (g) pH 8 in the presence of 0.1 mM Pb2+ at surface pressures of 10 mN/m (green), 20 mN/m (purple), 30 mN/m (blue), and 40 (red) mN/m; and calculated tilt and twist angles for the Rha−C18− C18 monolayer at (b) pH 2, (d) pH 5, (f) pH 8, and (h) pH 8 in the presence of 0.1 mM Pb2+ as a function of surface pressure.

The intensity ratio of the νas(CH2) and νs(CH2) bands in the experimental PM-IRRAS data can also be used to extract information about the twist of the hydrocarbon chain about its axis. The twist angle for an all-trans hydrocarbon chain is defined as the angle between the C−C−C plane and the plane formed by the chain axis and the surface normal and is defined by the following equation19

interfacial water molecules are replaced, resulting in negativegoing bands for the δ(H−O−H) and ν(O−H) modes, respectively. Due to spectral interference from these water bands, bands from Rha−C18−C18 in these regions are not useful for quantitative determination of molecular orientation. The two overlapping bands at ∼1735 cm−1 in the KBr transmission spectrum are from the ν(CO) modes of the ester and acid moieties in the headgroup. Unfortunately, these modes are very weak in the PM-IRRA spectrum, as shown in Figure 5, likely due to the confluence of several effects: only two CO moieties are present per molecule (compared with the 30 methylene moieties per molecule), the orientation of these CO moieties is not optimal for coupling with the surface electric field, and the number of Rha−C18−C18 molecules sampled in this experiment is relatively low due to the bulky size of the headgroup. Thus, these modes are not useful for further orientational analysis. In the spectral region from 3100 to 2800 cm−1, reflection− absorption bands arise from the νas(CH2) and νs(CH2) vibrations of the Rha−C18−C18 hydrocarbon chains. The ν(CH3) vibrations are typically weak and not usually observed.27 Since the two ν(CH2) bands are sensitive to molecular packing and, hence, molecular orientation, they are used to quantitatively elucidate values of the chain axis tilt angle. Recalling the surface selection rule at the air−water interface, the sign and intensity of the absorption bands in the PM-IRRA spectra are indicative of preferential orientation of the transition dipole moments.17,28 The νas(CH2) and νs(CH2) bands for Rha−C18−C18 are positive, indicating that their transition dipole moments are preferentially oriented largely parallel to the surface. Since these transition dipole moments are perpendicular to the hydrocarbon chain axis, the chain must be oriented largely along the surface normal. In order to determine the specific tilt angle of the chain axis, a quantitative comparison of the experimental data with the simulated PMIRRA spectra for Rha−C18−C18 is undertaken. Since the transition dipole moments of these two modes are mutually orthogonal to each other and to the hydrocarbon chain axis, the tilt angles of the transition dipole moments of these modes, θas and θs, and the tilt angle of the chain axis, θtilt, are related by29,30 cos2 θas + cos2 θs + cos2 θtilt = 1

⎡⎛ ⎞ ⎛ ⎞ ⎤1/2 A A ψ = arctan⎢⎜ as ⎟ ⎜ s ⎟ ⎥ ⎢⎣⎝ A s ⎠ ⎝ A as ⎠ ⎥⎦ film KBr

(16)

where (Aas/As)film is the integrated absorbance ratio of the νas(CH2) and νs(CH2) bands in the PM-IRRA spectrum from the Rha−C18−C18 monolayer on the water surface and (Aas/ As)KBr is the integrated absorbance ratio of the νas(CH2) and νs(CH2) bands in the transmission spectrum of a KBr pellet of Rha−C18−C18. It is also noted that, as a result of the tangent relationship, for a twist angle of 45°, also known as the trivial twist angle, no preferential twist orientation of the molecular chains exists.19 This trivial angle would be calculated, for example, for a collection of chains with a randomly distributed twist angle. Rha−C18−C18 Monolayers on Aqueous Subphases of Varying pH. At pH 2, the carboxylic acid group of Rha−C18− C18 is fully protonated and the molecule is nonionic. As shown in Figure 6a, the PM-IRRA spectra recorded at this pH exhibit two positive ν(CH2) bands with integrated absorbance values that increase as the monolayer is compressed from 10 to 40 mN/m. It can be seen qualitatively that the peak frequencies and widths of the νas(CH2) and νs(CH2) bands are sensitive to the packing of the surface Rha−C18−C18 molecules. More importantly, the integrated absorbance values, together with the simulated PM-IRRA spectra, provide quantitative orientation information about the Rha−C18−C18 alkyl chains that allows a complete picture of molecular orientation to be deduced. In the spectra shown in Figure 6a, the νas(CH2) and νs(CH2) bands are observed at 2917 and 2851 cm−1, respectively. These frequencies suggest a high degree of interchain coupling,31 and hence chain order, at the air−water interface at pH 2 due to favorable van der Waals interaction between the long hydrocarbon chains under conditions of close packing. The calculated θas and θs values are 52° and 50°, respectively, at a surface pressure of 10 mN/m; from these values, θtilt is determined using eq 15 to be 63° (Figure 6b). When the Rha−

(15) 4445

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C18−C18 monolayer is further compressed, the band intensities increase accordingly, an indication of a larger tilt of the transition dipole moments of the νas(CH2) and νs(CH2) modes. At the same time, the frequencies of both bands decrease, an indication that the alkyl chains become more ordered upon compression.31 When the surface pressure approaches its maximum achievable value of 40 mN/m before monolayer collapse, θas and θs and are calculated to be 62° and 50°, respectively, corresponding to a value of θtilt = 45°. Upon compression, therefore, the alkyl chains become less tilted (Figure 6b), although the absolute tilt angle is still relatively large compared to simpler surfactants such as fatty acids.19,29 For example, θtilt for stearic acid is ∼10°,29 a value significantly smaller than the tilt angle for Rha−C18−C18. Stearic acid is a much smaller molecule than Rha−C18−C18, possessing only one alkyl chain and a carboxylic acid headgroup. Indeed, the biggest contributor to the difference in alkyl chain orientation at the air−water interface for Rha− C18−C18 is the presence of the sugar moiety in its headgroup. The relatively large tilt angle of the alkyl chains for Rha−C18− C18 is proposed to be a result of the nature of the headgroup packing. It is further noted that, for surfactants with a headgroup larger in cross-sectional area than the tail, portions of the hydrocarbon chains near the headgroup are reported to be more ordered than those at the distal end of the chain.19,32 The surface pressure dependence of the twist angle of the Rha−C18−C18 chains (Figure 6b) suggests no statistically significant preferential twist angle at any surface pressure, since all values are close to the trivial angle. At a surface pressure of 10 mN/m, the twist angle is calculated to be ∼42°. At 20 mN/ m, the twist angle approaches the 45° trivial twist angle, and for further increases in surface pressure, the twist angle remains at about 45°, an indication of no preferential twist.19 The reversibility of Rha−C18−C18 intermolecular interactions, and hence the integrity of the monolayer during and after compression, was also evaluated. Often, when a monolayer is compressed sufficiently, it will curve at the interface and finally collapse into three-dimensional structures such as micelles or vesicles.33 This collapse is generally observed in the surface pressure−area isotherm as a rapid decrease in surface pressure. Interestingly, this phenomenon is not observed in surface pressure−area isotherms for Rha−C18− C18 at pH 2. To further explore this observation, PM-IRRA spectra were recorded for the Rha−C18−C18 monolayer at a surface pressure of 40 mN/m2 before further compression, after further compression beyond 40 mN/m2, and after subsequent decompression back to 40 mN/m2 as shown in Figure 7. Upon further compression beyond 40 mN/m2, the surface area between the trough barriers is reduced from 90 to 60 cm2. If parts of the monolayer collapsed to form solution aggregates in response to this decrease in surface area, the PM-IRRAS intensities would stay the same, since the molecular density at the air−water interface would be maintained essentially constant. However, if the monolayer collapsed to form stable multilayers at the water surface, the PM-IRRAS signal should increase, and the intensity increase should reflect the ratio of the surface areas before and after compression (before/after = 90 cm2/60 cm2 = 1.5). The experimental integrated absorbance ratio of the spectrum after compression to that before compression is ∼1.4, which supports the contention that multilayers are formed upon compression beyond the stable monolayer “solid state” at pH 2. After compression, the monolayer was decompressed back to a trough surface area of

Figure 7. PM-IRRA spectra from a Rha−C18−C18 monolayer at pH 2 at a surface pressure of 40 mN/m (green) in a trough surface area of 90 cm2, after further compression beyond 40 mN/m (red) to a trough surface area of 60 cm2, and after decompression (blue) back to a trough surface area of 90 cm2.

90 cm2 and the PM-IRRA spectrum was recorded. As shown in Figure 7, the absorbance values return to what they were before compression, indicating that collapse of the monolayer into a multilayer structure is a reversible process. At pH 5, the carboxylic acid of Rha−C18−C18 is partially deprotonated, and thus, the Rha−C18−C18 is a mixture of nonionic and anionic species. It might be expected that deprotonation and the presence of negative charge on the Rha−C18−C18 headgroup would result in a conformational change in the molecules in the monolayer. However, the experimental PM-IRRAS data shown in Figure 6c reveal no significant pH dependence of chain conformational order. The peak frequencies of the νas(CH2) and νs(CH2) bands are 2920 and 2850 cm−1, respectively, indicating alkyl chains that are highly ordered,31 even at the relatively low surface pressure of 10 mN/m. At this surface pressure, the tilt angle of the alkyl chain is determined to be 61° (Figure 6d) on the basis of the integrated absorbance values of these bands. As observed at pH 2, the tilt angle decreases as the surface pressure increases with a tilt angle of 44° at the maximum surface pressure of 40 mN/ m. The twist angle at pH 5 is again close to the trivial angle at all surface pressures.19 It increases slightly from 41° to 44° as the surface pressure increases from 10 to 20 mN/m. Beyond 20 mN/m, the twist angle stays constant at 44° (Figure 6d). The reversibility experiment was also executed at pH 5 (data not shown). In contrast to what was observed at pH 2, the results show that when reaching maximum surface pressure, the PM-IRRAS becomes independent of available surface area, suggesting that at this pH, the Rha−C18−C18 molecules prefer to enter solution as aggregates instead of forming multilayers at the air−water interface. This is attributed to the greater solubility of Rha−C18−C18 at pH 5 relative to pH 2. At pH 8, a value above the pKa of Rha−C18−C18, the carboxylic acid is completely deprotonated, making Rha−C18− C18 anionic.25 One might expect a change in alkyl chain packing in the monolayer at the air−water interface due to the negatively charged headgroup. However, the experimental PMIRRAS data shown in Figure 6e reveal no significant pH dependence of chain packing from what it was at pH 2 or 5. From energy-minimized molecular mechanics modeling and hydrogen−deuterium exchange studies using mass spectrometry reported elsewhere from this laboratory,34 we speculate that the rhamnolipid headgroup forms a compact pocket in which the anionic charge is buried along with its counterion. This 4446

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The compression experiment after reaching maximum surface pressure was also performed in presence of Pb2+ (data not shown). The results show that after reaching maximum surface pressure, the PM-IRRAS signal is dependent on the surface area after compression. Thus, the metal-complexed surfactants prefer to form multilayers at the air−water interface rather than enter into solution as aggregates. This is different from the behavior observed in the absence of Pb2+ at pH 8 but similar to the behavior of uncomplexed Rha−C18−C18 at pH 2. The similarity with the behavior at pH 2 is consistent with the limited solubility of the Rha−C18−C18−Pb2+ complex at pH 8 and in contrast to the solubility of the uncomplexed Rha− C18−C18 at pH 8. Molecular Modeling of Rha−C18−C18 at the Air− Water Interface. In general, the hydrophobic alkyl chains of surfactants tend to orient preferentially along the surface normal to maximize their packing density at the interface. However, Rha−C18−C18 has a tilt angle as large as 45° at the relatively high surface pressure of 40 mN/m. In order to rationalize this unusually large tilt angle, molecular modeling was employed to probe molecular packing at the air−water interface. The molecular structure of Rha−C18−C18 was energy-minimized using molecular mechanics in Spartan, as shown in Figure 8a. This structure shows that the rhamnose sugar and carboxylic acid moieties rotate toward each other to form a compact pocket structure, presumably due to intramolecular hydrogen bonding. In this structure, the rhamnose sugar is rotated in such a way that the methyl group points

headgroup configuration could isolate the charge and its counterion from neighboring headgroups, thereby minimizing electrostatic repulsion. Such minimization of repulsive interactions would allow little to no impact of headgroup charge on packing within the monolayer. On the basis of the peak frequencies of the νas(CH2) and νs(CH2) bands at pH 8 (Figure 6e), the alkyl chains appear to be highly ordered.31 At a surface pressure of 10 mN/m, the tilt angle of the alkyl chain is calculated to be 62° and decreases ∼5° for every increase of 10 mN/m in surface pressure to a minimum of 45° at the maximum surface pressure of 40 mN/m (Figure 6f). As at pH 2 and 5, the alkyl chain twist angle is again in the vicinity of the trivial angle for all surface pressures. The effect of further compression of the Rha−C18−C18 monolayer after reaching its maximum surface pressure was also investigated at pH 8 (data not shown). The results show that after reaching maximum surface pressure, the PM-IRRAS signal is independent of the surface area upon compression as observed at pH 5. This behavior suggests that the Rha−C18− C18 molecules prefer to enter solution as aggregates upon collapse instead of forming multilayers at the air−water interface due to their relatively high solubility at this pH. Rha−C18−C18 Monolayers on Aqueous Subphases Containing Pb2+. Rhamnolipids have been shown to readily form complexes with Pb2+ and therefore are of interest for the bioremediation of heavy metals from water and soil.35,36 Upon metal complexation, some degree of conformational change is expected in the rhamnolipid structure. To probe such conformational changes in Rha−C18−C18 monolayers at the air−water interface, PM-IRRAS is used here for water subphases at pH 8. The concentration of Pb2+ in the subphase was chosen to be 0.1 mM to ensure enough metal ions to force binding within the monolayer. The surface pressure−area isotherm in the presence of Pb2+ in the subphase is shown in the Supporting Information (Figure S5). PM-IRRA spectra for the Rha−C18−C18 monolayers on subphases containing 0.1 mM Pb2+ at different surface pressures are shown in Figure 6g. The spectra exhibit a slight decrease in peak frequency for the νas(CH2) band with pressure from 2922 to 2919 cm−1 and for the νs(CH2) band from 2852 to 2850 cm−1. This decrease of peak frequency is an indication of a slightly higher degree of chain order with higher pressure. An increase in peak absorbance is also observed as the surface pressure of the monolayer increases. The positive-going bands for the νas(CH2) and νs(CH2) modes indicate a preferential orientation of the transition dipole moments parallel to the surface at all surface pressures. The tilt angle at a surface pressure of 10 mN/m is calculated to be 53° (Figure 6h), a value 9° lower than that observed in the absence of Pb2+. The tilt angle decreases to 36° as the surface pressure is increased to 40 mN/m, a value that is similarly 9° lower than that observed in the absence of Pb2+. The twist angle of the alkyl chain is consistently near the trivial angle for all surface pressures (Figure 6h), indicating no preferred twist angle of the alkyl chains. This behavior is consistent with the complexed Rha−C18−C18 having a hydrophilic headgroup with a cross-sectional area larger than the sum of the areas of the two hydrocarbon chains. Collectively, these results suggest that the structure of the Rha−C18−C18−Pb2+ complex in the monolayer film is similar, although not identical, to that of the uncomplexed Rha−C18− C18 monolayer.

Figure 8. (a) Energy-minimized molecular mechanics structure of Rha−C18−C18 computed in Spartan in side view, front view, and top view. (b) Optimal molecular packing of a Rha−C18−C18 monolayer at the air−water interface with the hydrophilic headgroups oriented largely perpendicular to the surface normal and the alkyl chains tightly packed at their van der Waals radii. 4447

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toward the hydrophobic alkyl chains and away from the hydrophilic headgroup. On the basis of this three-dimensional structure, the Rha−C18−C18 possesses an overall cylindrical shape in front view and a boot shape in side view. The top view clearly shows that the Rha−C18−C18 hydrophilic headgroup has a larger cross-sectional area than the sum of the areas of its two hydrophobic alkyl chains. On the basis of this three-dimensional structure, it appears that the factor critical in dictating molecular packing of Rha− C18−C18 at the air−water interface is its boot shape. In order to pack the Rha−C18−C18 molecules tightly at the air−water interface, the large hydrophilic headgroup must be packed in such a fashion that maximum surfactant density can be achieved. If the alkyl chains are oriented along the surface normal, the Rha−C18−C18 hydrophilic headgroups would be oriented largely parallel to the interface, requiring a large crosssectional area within the monolayer. Moreover, in this arrangement, as a result of the large cross-sectional area of the headgroup, the alkyl chains would be relatively far apart and would not attain good van der Waals packing. However, if the hydrophilic headgroup is oriented more perpendicular to the interface, as shown in Figure 8b, its cross-sectional area is minimized while allowing the Rha−C18−C18 alkyl chains to be tightly packed, thereby maximizing the Rha−C18−C18 number density at the air−water interface. One additional advantage of this orientation is that the rhamnose moieties from different Rha−C18−C18 molecules within the monolayer are oriented more or less parallel to each other, possibly allowing additional hydrogen-bonding interactions that further stabilize the structure. Headgroup−headgroup hydrogen bonding between surfactant molecules at the air−water interface similar to what is envisioned to occur here has been reported previously by Vico et al. for monoacylated βcyclodextrin monolayers.37 In the presence of Pb2+, rhamnolipids are thought to form complexes by incorporating the metal cation into or partially into the binding pocket wherein it can associate with multiple oxygen atoms along the headgroup backbone.34 Within this binding pocket, two of the oxygen atoms are hydroxyls on the rhamnose sugar moiety at carbon atoms 2 (C2) and 3 (C3). Other oxygen atoms that are thought to be involved in binding are from the carboxylic acid group. Taking this coordination picture into consideration, the complex of Rha−C18−C18 and Pb2+ in a 1:1 ratio was energy-minimized using molecular mechanics in Spartan. The resulting energy-minimized structure of the Rha−C18−C18−Pb2+ complex is compared to that of the uncomplexed Rha−C18−C18 in Figure 9. The angle between the axis of the hydrocarbon chains and the axis of the elliptical headgroup is ∼113° for free Rha−C18−C18 but ∼120° for the Rha−C18−C18−Pb2+ complex. This angular difference of 7° allows the alkyl chains to be less tilted by ∼7° to attain optimal van der Waals packing. This calculated value corresponds quite well to the experimentally observed tilt angle decrease of 9° in going from the free Rha−C18−C18 to the Rha−C18−C18−Pb2+ complex. Thus, the results of molecular modeling substantiate the conclusions about orientation made on the basis of the PM-IRRAS data.

Figure 9. Energy-minimized molecular mechanics structures of the free Rha−C18−C18 and a Rha−C18−C18−Pb2+ complex; the presence of Pb2+ in binding pocket causes 7° distortion of angle between alkyl chain axis and headgroup axis.

pH, consistent with what has been observed previously.38 PMIRRA spectra indicate an increase in alkyl chain order and a decrease in alkyl chain tilt angle as the surface pressure of the monolayer increases. Although a change in pH changes the protonation state of the carboxylic acid headgroup, the alkyl chain tilt angle shows virtually no dependence on pH, suggesting little conformational change of the headgroup upon deprotonation, and hence, similar molecular orientation and packing within the monolayer. This is rationalized by the compact pocket structure of the rhamnolipid headgroup that largely shields the negative charge. In this way, the character of the negative charge is diminished such that the electrostatic repulsion is negligible. The large tilt angle of the alkyl chains caused by the size of the headgroup and the need for the chains to tilt in order to achieve tight packing is supported by molecular modeling. The system energy may be further lowered by hydrogen bonding between the headgroups that is facilitated by this orientation. The presence of the metal cation Pb2+ decreases the tilt angle of the chains by ∼9° by formation of rhamnolipid−Pb2+ complexes within the monolayer. Molecular modeling based on the previously proposed structure of the complex with the Pb2+ partially within the binding pocket substantiates the observed decrease in alkyl chain tilt angle. The experimental data also suggest that, at pH 2 or in presence of Pb2+, multilayers can form at the air−water interface beyond the surface pressure of monolayer collapse instead of the formation of micelles in solution. However, at pH 5 and 8, at which the solubility of the free Rha−C18−C18

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CONCLUSIONS PM-IRRAS was used to study Rha−C18−C18 molecular orientation in monolayers at the air−water interface. The results suggest that the tilt angle of the hydrocarbon chain is dependent on the surface pressure but independent of subphase 4448

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increases substantially, only monolayers are formed at the air− water interface. Deprotonation increases the hydrophilicity of Rha−C18−C18, making formation of solution micelles easier.9

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ASSOCIATED CONTENT

S Supporting Information *

Description of the synthesis of Rha−C18−C18, a plot of the zero-order Bessel function as a function of frequency, a description of the Kramers−Kronig transformation used to extract n and k values for Rha−C18−C18 from the transmission FTIR spectrum, and the surface pressure−area isotherms for Rha−C18−C18 above subphases of pH 2, 5, and 8, as well as a subphase of pH 8 containing 0.1 mM Pb2+. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Address †

Department of Chemistry, The Ohio State University, Columbus, OH.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge support of this research by the National Science Foundation award CHE-0714245. Partial support for A.M. and for the FTIR instrumentation for this study came from the National Science Foundation award CHE0848624.

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