Determination of the Acid Dissociation Constant of the Biosurfactant

The acid dissociation constant in water for a monorhamnolipid mixture extracted from Pseudomonas aeruginosa ATCC 9027 has been determined using ...
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Anal. Chem. 2006, 78, 7649-7658

Determination of the Acid Dissociation Constant of the Biosurfactant Monorhamnolipid in Aqueous Solution by Potentiometric and Spectroscopic Methods Ariel Lebro´n-Paler and Jeanne E. Pemberton*

Department of Chemistry, University of Arizona, 1306 East University Boulevard, Tucson, Arizona 85721 Bridget A. Becker,† William H. Otto,‡ and Cynthia K. Larive†

Department of Chemistry, University of Kansas, Lawrence, Kansas 66045 Raina M. Maier

Department of Soil, Water and Environmental Science, University of Arizona, Tucson, Arizona 85721

The acid dissociation constant in water for a monorhamnolipid mixture extracted from Pseudomonas aeruginosa ATCC 9027 has been determined using potentiometry and two spectroscopic approaches at concentrations below and above the critical micelle concentration (cmc). Potentiometric titrations resulted in pKa values ranging from 4.28 ( 0.16 to 5.50 ( 0.06 depending on concentration. 1H NMR spectrochemical titrations at concentrations below the cmc revealed a pKa value of 4.39 ( 0.06. ATR-FT-IR spectrochemical titrations on solutions well above the cmc gave a pKa value of 4.84 ( 0.05. The value of 4.28 for the free rhamnolipid molecule for concentrations below the cmc differs markedly from that reported previously. However, the pKa of 5.50 for surface-adsorbed and solution aggregates correlates closely to that previously reported. Differences in these pKa values are rationalized in terms of the pH- and concentrationdependent aggregation behavior of rhamnolipids in aqueous solution. An interesting but complex class of molecules known as rhamnolipids has been proposed for bioremediation of organic (e.g., polycyclic aromatic hydrocarbons and polychlorinated biphenyls) and heavy metal (e.g., Cd2+, Pb2+) contaminants.1-3 These materials are also being actively explored for use as * To whom correspondence should be addressed. E-mail: pembertn@ u.arizona.edu. Phone: 520-621-8245. † Current address: Department of Chemistry, University of Californias Riverside, Riverside, CA 92521. ‡ Current address: Department of Chemistry, University of Maine at Machias, Machias, ME 04654. (1) Bai, G.; Brusseau, M. L.; Miller, R. M. J. Contam. Hydrol. 1998, 30, 265279. (2) Ochoa-Loza, F. J.; Artiola, J. F.; Maier, R. M. J. Environ. Qual. 2001, 30, 479-485. (3) Mata-Sandoval, J. C.; Karns, J.; Torrents, A. Environ. Sci. Technol. 2000, 34, 4923-4930. 10.1021/ac0608826 CCC: $33.50 Published on Web 10/18/2006

© 2006 American Chemical Society

antimicrobial and antifungal agents,4-6 as components in topical pharmaceutical preparations,7,8 and as additives to food and cosmetics.9,10 Rhamnolipids are glycolipid biosurfactants consisting of one or more rhamnose sugar headgroups11,12 and one or more aliphatic chain tail groups. They are produced by strains of Pseudomonas aeruginosa and were first characterized by Jarvis and Johnson in 1949.12-14 Rhamnolipids produced by P. aeruginosa are composed of two alkyl chains and either one11 or two12 rhamnose sugar units. Those rhamnolipids with a single rhamnose sugar group and alkyl chain tails ranging in length from pentyl to nonyl are called monorhamnolipids, while those with two rhamnose units and alkyl chains ranging from pentyl to nonyl are called dirhamnolipids. The structure for a monorhamnolipid with two heptyl chains is shown in Scheme 1. Rhamnolipids are weak acids due to the presence of the carboxylic acid moiety and are known to undergo aggregation in solution. Values of the critical micelle concentration (cmc) for rhamnolipids are dependent on chemical environment and have been reported by different investigators to range from ∼10 to 120 µM.15-18 At concentrations above the cmc, rhamnolipids form (4) Benincasa, M.; Abalos, A.; Oliveira, I.; Manresa, A. Antonie van Leeuwenhoek 2004, 85, 1-8. (5) Haba, E.; Abalos, A.; Jauregui, O.; Espuny, M. J.; Manresa, A. J. Surfactants Deterg. 2003, 6, 155-161. (6) Abalos, A.; Pinazo, A.; Infante, M. R.; Casals, M.; Garcia, F.; Manresa, A. Langmuir 2001, 17, 1367-1371. (7) Piljac, G.; Piljac, V. U.S. Patent 5455232, 1995. (8) Stipcevic, T.; Piljac, T.; Isseroff, R. R. J. Dermatol. Sci. 2005, 40, 141-143. (9) Lang, S.; Wullbrandt, D. Appl. Microbiol. Biotechnol. 1999, 51, 22-32. (10) Maier, R. M.; Soberon-Chavez, G. Appl. Microbiol. Biotechnol. 2000, 54, 625-633. (11) Rendell, N. B.; Taylor, G. W.; Somerville, M.; Todd, H.; Wilson, R.; Cole, P. J. Biochim. Biophys. Acta 1990, 1045, 189-193. (12) Jarvis, F. G.; Johnson, M. J. J. Am. Chem. Soc. 1949, 71, 4124-4126. (13) Torrens, J. L.; Herman, D. C.; Miller-Maier, R. M. Environ. Sci. Technol. 1998, 32, 776-781. (14) Zhang, Y.; Miller, R. M. Appl. Environ. Microbiol. 1992, 58, 3276-3282. (15) Champion, J. T.; Gilkey, J. C.; Lamparski, H.; Retterer, J.; Miller, R. M. J. Colloid Interface Sci. 1995, 170, 569-574.

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Scheme 1

micelles, vesicles, or lamella depending on solution pH, concentration, and presence of electrolytes. The strongly surface-active behavior of rhamnolipids, the presence of multiple rhamnolipid congeners, and the dependence of rhamnolipid aggregate morphology on solution pH make precise determination of pKa values for these systems using conventional potentiometric methods difficult. Although it has been widely assumed that rhamnolipids behave as simple carboxylic acids in solution, only one report of a pKa value based on potentiometric determination for any rhamnolipid has been published,16 and the pKa value of 5.6 reported is ∼1.5 pK units above that expected for a simple carboxylic acid. This value presumably resulted from titration of a rhamnolipid solution in the millimolar concentration range, but the claim was not substantiated with published titration data. Indeed, this previously reported value corresponds to the pH boundary at which rhamnolipid aggregates undergo a transition from the lamellar phase to vesicles. Since this work was published, an increasing number of studies have appeared in which results are interpreted by reliance on this pKa value without careful consideration of the level and state of solution aggregation of the rhamnolipid biosurfactant at different concentrations.19,20 Given the central role of aggregation in the performance of rhamnolipids in different applications, understanding the dependence of acid dissociation on pH and aggregation state in these systems is critical. Kanicky and Shah have used traditional potentiometric titration to evaluate the effect of molecular aggregation on dissociation constant for various fatty acids containing carboxylic acid groups.21 These researchers showed that hydrophobic chain length of the surfactant affects molecular packing when the chain is longer than ∼6 carbons.21 The pKa increases accordingly from the typical carboxylic acid value of ∼4.8 due to increased interactions between premicellar concentrations of individual anionic surfactant molecules with concomitant stabilization of the acid proton as a means to minimize electrostatic repulsion. Similar results have been observed for dissociation of surface-adsorbed carboxylic acids as well.22,23 (16) Ishigami, Y.; Gama, Y.; Nagahora, H.; Yamaguchi, M.; Nakahara, H.; Kamata, T. Chem. Lett. 1987, 763-766. (17) Hung, H. C.; Shreve, G. S. J. Phys. Chem. B 2001, 105, 12596-12600. (18) Garcia-Junco, M.; De Olmedo, E.; Ortega-Calvo, J. J. Mol. Microbiol. 2001, 3, 561-569. (19) Helvaci, S. S.; Pekere, S.; Ozdemir, G. Colloids Surf., B 2004, 35, 225233. (20) Ozdemir, G.; Peker, S.; Helvaci, S. S. Colloids Surf., A 2004, 234, 135143. (21) Kanicky, J. R.; Shah, D. O. Langmuir 2003, 19, 2034-2038. (22) Bard, A. J.; Hu, K. Langmuir 1997, 13, 5114-5119.

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Similar behavior is also expected for carboxylic acid surfactant concentrations far above the cmc, as in the case of the potentiometric titration of rhamnolipid performed by Ishigami et al.16 In light of such aggregation, the high value of the pKa reported by these researchers might be correct. Ideally, however, the pKa would be determined at solution concentrations below the cmc in order to obtain the true molecular rhamnolipid pKa. Under these conditions, long-range van der Waals interactions between hydrophobic chains are relatively weak and individual monomers predominate. However, several groups have also reported the existence of premicellar aggregation of acidic surfactants, such as the formation of dimers and oligomers, at concentrations below but close to the monomeric surfactant cmc;24,25 the influence of these interactions should also be taken into consideration in determination of the pKa. Finally, Kanicky and Shah have also shown that the presence of lipids of different chain length reduces surfactant cooperativity, resulting in more loosely packed molecular aggregates and lower pKa values.21 This offers an advantage for the heterogeneous rhamnolipid system of interest here, since the pKa determination can be done at concentrations close to the cmc with a reduced impact of aggregation on the rhamnolipid deprotonation process. The goal of the work reported here was to determine the pKa of highly purified monorhamnolipid (hereafter referred to as rhamnolipid) from P. aeruginosa ATCC 9027 using potentiometric titration and 1H NMR and ATR-FT-IR spectrochemical titrations. Given the difficulties associated with conventional potentiometric titrations of aggregating and strongly adsorbing species, the molecular information content of the NMR and FT-IR spectroscopies allows determination of rhamnolipid pKa with greater specificity. EXPERIMENTAL SECTION Rhamnolipid Purification. Rhamnolipid extracted from P. aeruginosa ATCC 9027 was partially purified according to previous reports.14 This partially purified material was further purified using an extensive method developed in this laboratory for larger scale purification.26 Briefly, ∼1.8 g of the monorhamnolipid mixture is loaded onto a 22 × 300 mm gravity-based glass chromatography column packed with 45 g of 60-Å-pore silica gel. A solvent mixture of hexane, dichloromethane, ethyl acetate, chloroform, and (23) Fernandez, M. S. F.; Fromherz, P. J. Phys. Chem. 1977, 81, 1755-1761. (24) Wangnerud, P.; Jonsson, B. Langmuir 1994, 10, 3542-3549. (25) Zana, R. J. Colloid Interface Sci. 2002, 246, 182-190. (26) Lebro´n-Paler, A.; Pemberton, J. E.; Maier, R. M.; Becker, B.; Larive, C. K., to be submitted.

methanol in the ratio (v/v) 6:6:6:1:1 with 0.1% acetic acid is used to slowly elute the biosurfactant and remove the majority of the pigments. Collection of purified monorhamnolipid is initiated in fractions exhibiting positive rhamnose confirmation with anthrone reagent dissolved in H2SO4. Solvent is removed by rotoevaporation and the monorhamnolipid resuspended in water at pH ∼7-8 prior to lyophilization. Biosurfactant so purified was used in the potentiometric and ATR-FT-IR spectrometric titration experiments. For the NMR experiments, a faster purification method was developed. This purification starts by dissolving up to 100 mg of the rhamnolipid in water and adjusting the solution pH to 8 with NaOH. This aqueous solution is then loaded onto a 3-mL C18 solidphase extraction cartridge (Waters Sep-Pak) and rinsed with 3 mL of water and then 10% aqueous methanol until the remaining purple-brown pigment is removed by visual assessment. The rhamnolipid is then eluted with 4 mL of 90% methanol. The majority of the methanol is removed by blowing N2 gas over the vial containing the eluted solution in a 35 °C water bath. A 1-mL aliquot of water was added to the residual material prior to lyophilizing. In the final step from both purifications, the rhamnolipid is lyophilized overnight using a Labconco Lymph Lock 6 freeze-drier. The lyophilized powder was stored at -20 °C. Dynamic Light Scattering Measurements. Solutions for dynamic light scattering measurements were prepared by dissolving lyophilized monorhamnolipid in filtered, deionized water. Solutions containing 1 mM rhamnolipid were adjusted to pH 7.17 or 3.20 using either NaOH or HCl. The solutions were equilibrated for 16 h prior to measurement. The light scattering experiments were performed at room temperature using a Brookhaven instrument and 9000AT autocorrelator. Scattered light was detected with an EMI 9863 photomultiplier tube mounted on a BI-200M goniometer at 90° to incident radiation of 632 nm from a 50-mW HeNe laser (JDS Uniphase). Potentiometric Rhamnolipid Titration. Aqueous solutions deprived of CO2 by boiling and containing purified 5, 50, or 300 µM rhamnolipid mixture were prepared in Nalgene volumetric flasks with the ionic strength fixed at 0.1 M with NaCl. Although rhamnolipid species of different chain lengths are present in the mixture, the solution concentrations were calculated using the molecular weight of the main species C26H48O9 (FW 504). These concentrations were chosen to represent values below and above the cmc, respectively, determined for this rhamnolipid preparation to be 10 µM under the solution conditions used for the potentiometric titration.26 The addition of NaCl to fix the ionic strength was necessary to obtain stable pH readings at this low concentration and to minimize errors associated with the electrode calibration, liquid junction potential of the reference electrode, and stirring.27-29 Titrations were performed under a N2-saturated water vapor environment at room temperature using a ThermoOrion model 91-16 semimicro-combination pH-Ag/AgCl electrode attached to a ThermoOrion model 710 pH/ISE meter. Calibration was performed with commercial pH buffers 4.00, 7.00, and 10.00 (VWR International). NaOH titrant was standardized with primary standard potassium hydrogen phthalate (Fisher Scientific, 100.02%.) (27) Davison, W.; Woof, C. Anal. Chem. 1985, 57, 2567-2570. (28) Covington, A. K.; Whalley, P. D.; Davison, W. Anal. Chim. Acta 1985, 169, 221-229. (29) Brandariz, I.; Vilarino, T.; Alonso, P.; Herrero, R.; Fiol, S.; Vicente, M. E. S. d. Talanta 1998, 46, 1469-1477.

For rhamnolipid titrations, pH readings were acquired 20 s after adding the titrant, when the pH response stabilized. The end point was obtained from the first derivative of the resulting titration curve of pH as a function of fractional equivalent volume added, f, as the point where the slope of the titration curve, d(pH)/df, is the greatest or the fractional equivalent volume added at which the second derivative is 0. The pKa was determined as the pH at 0.5f. The reliability of this potentiometric method for such low concentrations of rhamnolipid was tested by titrating a 50 µM solution of potassium hydrogen phthalate under the same experimental conditions. For the second ionizable proton, the pKa was determined to be 5.35 ( 0.05, in good agreement with the published value of 5.4030 although lower values down to 4.95 have been reported in 0.1 M NaCl.29,31 The titration curve for this system is shown as Figure S1 in Supporting Information. 1H NMR Rhamnolipid Titration. All rhamnolipid and indicator solutions were prepared in 90% H2O/10% D2O. Because the solutions contained only 10% D2O, no correction was made to compensate for the deuterium isotope effect.32,33 The intense solvent resonance was suppressed using presaturation through a long selective pulse on water prior to the hard pulse.34 For titration of the indicator species, 2 mM solutions of imidazole (Fisher Scientific), potassium hydrogen phthalate (Sigma), and NH4(HCO2) (Sigma) were prepared. The chemical shift reference agent, tert-butyl alcohol, was added at a concentration of ∼1 mM. The ionic strength was held constant by preparing all solutions in 0.2 M KCl. pH values of indicator solutions were measured with potentiometry using a 3.5 × 183 mm combination glass electrode (Cypress Systems) as the solution pH was raised from 1.63 to 8.21 in ∼0.2 pH unit steps. Two additional data points were measured at pH 8.98 and 9.88 to complete the titration of imidazole. For the rhamnolipid titration, solutions were prepared to contain ∼50 µM rhamnolipid, 200 µM concentrations of the three pH indicators, and 1 mM tert-butyl alcohol. The solution pH was adjusted by additions of aliquots of NaOD or DCl diluted with protonated water. 1H NMR measurements were performed with a Varian 600MHz NMR spectrometer. All spectra were referenced to tert-butyl alcohol using a chemical shift of 1.255 ppm as measured against 3-(trimethylsilyl)propionate. The chemical shifts were measured in hertz and converted to ppm to yield the maximum number of significant figures. Chemical shift versus pH data for the monoprotic (formic acid and imidazole) and diprotic (phthalic acid) titration indicators were fit using MicroMath Scientist for Windows (version 2.01). The resulting parametrized equations were used to calculate the pH of the rhamnolipid solutions. When the chemical shifts of the acid or base forms of the indicator could be accurately determined from the 1H NMR spectra, these parameters were fixed in the Scientist fits. However, for some indicators and for the acidic form of the rhamnolipid, the limiting chemical shifts could not be measured directly and were fit in Scientist along with the Ka. (30) Harris, D. C., Quantitative Chemical Analysis; W. H. Freeman and Co.: New York, 2001. (31) Rey-Castro, C.; Castro-Varela, R.; Herrero, R.; Vicente, M. E. S. d. Talanta 2003, 60, 93-101. (32) Baucke, F. G. K. J. Phys. Chem. B 1998, 102, 4835-4841. (33) Schowen, B. K.; Schowen, R. L. Methods Enzymol. 1982, 87, 551-606. (34) Prestegard, J. H. In Methods in 1D & 2D Liquid-Phase NMR; Academic Press: New York, 1988; pp 435-488.

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ATR-FT-IR Rhamnolipid Titration. Rhamnolipid solutions were prepared by dissolving the purified rhamnolipid in 18 MΩ Millipore Milli-Q, UV-treated water (Millipore Corp.) The pH was adjusted with dilute NaOH (Aldrich, 99.99%) or HCl (Mallinkcrodt, reagent grade) solution. pH was measured at ambient temperature with a ThermoOrion Ross Sure Flow model 8175 pH electrode attached to a ThermoOrion model 710 pH/ISE meter. The final concentration of each sample was maintained at 300 µM. Less than 1 mL/sample was needed for the ATR-FT-IR measurements. ATR-FT-IR spectra were acquired in a Magna-IR model 550 spectrometer equipped with a liquid N2-cooled MCT-A detector. A Harrick Horizon multiple internal reflection accessory (Harrick Scientific) was used for the ATR measurements. During the spectrochemical titration, the accessory was kept under a closed N2 environment to eliminate water vapor from the IR beam pathway, to improve the spectral quality at low concentrations and also to prevent the CO2 equilibrium with water from affecting the solution pH during the measurements. The internal reflection element (IRE) used was a Harrick ZnSe 50 × 10 × 2 mm 45° ATR trapezoid optical crystal. These dimensions allow 13 reflections inside the reflectance medium and a depth of penetration of 350 nm calculated at 1720 cm-1. The effective path length of this IRE is 1480 nm. ATR-FT-IR spectra were acquired using 500 coadded scans of both sample and reference at 4-cm-1 resolution with Happ-Genzel apodization. The spectral region between 1500 and 1800 cm-1 was baseline corrected and analyzed with GRAMS/32 software (version 5.21, ThermoElectron Corp). The peak-fitting application in GRAMS/ 32 is based on the widely used Levenberg-Marquardt method of nonlinear least-squares fitting for decomposition of overlapping peaks. The iteration solution is considered to have converged when the reduced χ2 was minimized and the calculated peak envelope closely matched the experimental peak envelope. A 50: 50 mixture of Gaussian and Lorentzian line shapes was used for peak fitting, as it fit the data more closely than either pure Gaussian or pure Lorentzian models. This algorithm has proven to be effective and accurate in previous work from this laboratory.35 The integrated molar absorptivity of the spectral envelope containing the ν(CdO) modes of the ester carbonyl and carboxylic acid groups was determined by transmission FT-IR. Rhamnolipid solutions from 1 to 10 mM in D2O (Cambridge Isotope Laboratories, 99.9%) were injected into a ThermoNicolet/Spectra Tech demountable liquid cell with two CaF2 windows and a path length of 150 µm.36 Spectra were acquired using 100 coadded scans of both sample and D2O reference at 4-cm-1 resolution with HappGenzel apodization. A value for  of 14 703 M-1 cm-2 was determined from the slope of a linear Beer’s law plot constructed from the integrated absorbance of the broad band in the 16801760-cm-1 range as a function of rhamnolipid concentration. RESULTS AND DISCUSSION Acid-Base and Aggregation Characteristics of Rhamnolipid. Rhamnolipid obtained from purification of the native P. aeruginosa colony mother liquor is a mixture of monorhamnolipid molecules. As elaborated elsewhere,26 although most individual molecular species of this mixture have been identified, some (35) Pasilis, S. P.; Pemberton, J. E. Inorg. Chem. 2003, 42, 6793-6800. (36) Jang, W. H.; Miller, J. D. Langmuir 1993, 9, 3159-3165.

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Figure 1. Dynamic light scattering for aqueous 1 mM monorhamnolipid solutions at pH 3.2 (s) and pH 7.17 (‚‚‚).

components remain unknown. In the presence of multiple molecular forms, measurements on this mixture represent a weighted average of the characteristics of the individual molecular components. Thus, the pKa of the carboxylic acid moiety determined from the efforts reported here will represent the weighted average of the mixture. Among the monorhamnolipid forms, known molecular differences are restricted to alkyl chain length and degree of saturation26 that may result in a range of pKa values for specific molecular forms.21 Thus, until progress is made in the isolation of tractable quantities of these different molecular forms for further experimental study, the pKa is treated here as a single constant while remaining cognizant that it is a weighted average of the rhamnolipid mixture. Nevertheless, due to the weak acid characteristics of rhamnolipid species, their aggregation is a sensitive function of solution pH. Rhamnolipids are known to form lamellar phases at low pH values where the carboxylic acid is protonated; these then transform into vesicles and then to micelles as the pH is raised and the carboxylic acid becomes deprotonated.15 Dynamic light scattering results at pH values on both sides of the region that is expected to contain the pKa for the rhamnolipid are shown in Figure 1. In a solution of 1 mM rhamnolipid at pH 7.17 (dashed line in Figure 1), well-defined monodisperse rhamnolipid spherical aggregates are formed15 with an average diameter of 270 nm. For a rhamnolipid solution of identical concentration at pH 3.2 (solid line in Figure 1), the aggregates are considerably larger, with an average diameter of 2500 nm, and polydisperse, as indicated by the breadth of the light scattering response. The presence of these large aggregates at low pH must be considered in interpreting the results from the three techniques used in the pKa analysis described below. Potentiometric Determination of pKa. The potentiometric pH titration of rhamnolipid solutions was performed at one concentration below the cmc, 5 µM, and two concentrations above the monorhamnolipid cmc, 50 and 300 µM. The effect of low ionic strength on potentiometric analysis of dilute systems using conventional pH electrodes is well documented. Systematic error can be minimized in such analyses by performing titrations at relatively high ionic strength; these titrations were therefore performed at an ionic strength of 0.1 M. However, changes in

solution ionic strength are known to alter the aggregation behavior of surfactants. For this rhamnolipid, increasing ionic strength results in lowering of the cmc from slightly greater than 100 µM at low ionic strengths (e.g., 0.001 M) to ∼10 µM at high ionic strengths (e.g., 0.1 M).26 As a result, the titration of premicellar concentrations of rhamnolipid is performed close to the limit for reliable potentiometric results. The acid concentration limit at which a reliable inflection point can be observed by potentiometric titration has been theoretically predicted to be CHA > (32 Kw)1/2 by Roller.37,38 Our lowest concentration of 5 µM is 1 order of magnitude above this limit, thus generating confidence in its validity. Figure 2 shows the resulting pH titration curves for these solutions as a function of fractional equivalent volume added, f, and the first derivative of this curve, d(pH)/df. At rhamnolipid concentrations of 5 and 50 µM (Figure 2a and b, respectively), the pKa values determined are 4.36 ( 0.07 and 4.28 ( 0.16, respectively. For the 300 µM solution, this pKa increases to 5.50 ( 0.06, similar to the value determined by Ishigami et al.16 It is interesting to note the features that appear in the titration curves as the concentration of rhamnolipid increases. The titration of a 5 µM solution shows the characteristic sigmoidal shape expected for a pure monoprotic acid, with only one clearly-defined inflection point (consistent with a single peak in the first-derivative curve.) However, for titration of a 50 µM solution, the titration curve deviates from the expected behavior in that the increase in pH begins considerably before the volume at which half of the rhamnolipid is deprotonated. As a result, the first derivative reproducibly indicates what appear to be multiple end points before the main one. The situation becomes even more complex for titration of the 300 µM solution in which the addition of titrant causes an almost linear pH change until a sharp inflection point is seen at the end point. This change in pH prior to the main end point in solutions above the cmc is not the result of kinetic complications due to slow pH stabilization in these complex media. After the addition of each titrant aliquot, the solution approaches its final pH and reproducibly stabilizes after ∼20 s. Thus, these changes must be associated with the deprotonation of rhamnolipids in distinct chemical environments. For example, in the 300 µM solution, the linear pH change occurs in solutions that are visibly cloudy, suggesting the presence of large rhamnolipid aggregates, before becoming clear for solution pH values beyond the end point. Moreover, as has been observed in previous studies using transmission electron microscopy,15 the aggregates are polydisperse in size at low pH. This polydispersity in aggregate size creates a diversity of carboxylic acid chemical environments due to different degrees of surface curvature that alter the acid dissociation constant. Thus, the large shift in pKa to higher values for this system is thought to reflect a response dominated by the acid dissociation of aggregated rhamnolipids, both in solution and on the surface of the glass pH electrode. 1H NMR Spectrochemical Determination of pK . Given the a complexities associated with potentiometric titration of the rhamnolipid, alternate analytical techniques were explored in an attempt to validate further the potentiometric results. For a weak acid such as monorhamnolipid, the NMR chemical shifts of protons close (37) Roller, P. S. J. Am. Chem. Soc. 1932, 54, 3485-3499. (38) Laitinen, H. L.; Harris, W. E., Chemical Analysis, 2nd ed.; McGraw-Hill: New York, 1975; p 43.

Figure 2. Potentiometric pH titration curves (- ‚ - ) and their first derivatives (‚‚‚) for rhamnolipid in 0.1 M NaCl (a) 5, (b) 50, and (c) 300 µM.

to the protonation site are pH sensitive, because the local electron density around these nuclei changes as the protonation state of the functional group changes. Furthermore, 1H NMR chemical shift indicators can be used to provide an independent measure of solution pH throughout the course of the titration, thereby improving the precision of the pKa value determined with this method. This is an important advantage for rhamnolipid solutions with their considerable chemical complexity. It should be noted, however, that although NMR spectra can be measured on rhamnolipid solutions close in concentration to the cmc, these measurements are limited at low pH by the reduced solubility Analytical Chemistry, Vol. 78, No. 22, November 15, 2006

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and line broadening that accompany rhamnolipid aggregation as the charge on the carboxylate group is neutralized. As a result, only part of the titration curve can be obtained. From rhamnolipid acid dissociation in aqueous solution (Scheme 1), assuming an activity coefficient of unity, relationships can be derived that describe the titration curves in terms of the NMR chemical shift as a function of pH. In aqueous solution, proton transfer is fast on the NMR time scale, and the observed chemical shift (δobs) of an affected nucleus is a weighted average of the chemical shift of the acid form (δHA) and the chemical shift of the conjugate base form of the molecule (δA-):

δobs ) RHAδHA + RA-δA-

(1)

where the weighting factors (RHA, RA-) are the fractions of the acid and conjugate base species, respectively.39,40 These fractions, RHA and RA-, can be expressed as a function of Ka for the weak acid and the proton concentration, [H+].

RHA )

[HA] [H+] ) + ; CHA [H ] + K a

RA- )

Ka [A-] ) + CHA [H ] + K

(2) a

Substituting into eq 1 gives an expression for the observed chemical shift, δobs:

δobs )

(δHA[H+]) + (δA- Ka) Ka + [H+]

(3)

Using eq 3, it is possible to determine the pKa of a molecule by measuring the chemical shifts of nuclei (in this case 1H) near the acidic functional group as the solution pH is varied. Similarly, a molecule can be used as an internal NMR pH indicator by establishing the relationship between chemical shift and solution pH. Figure 3 shows the 1H NMR spectrum of a pH 7.42 aqueous solution containing 50 µM rhamnolipid and the indicators phthalic acid (P), imidazole (I), and formic acid (F). The rhamnolipid 1H resonances are observed between 0.5 and 5.5 ppm. The molecules selected as pH indicators have resonances in the aromatic region of the spectrum and, therefore, do not interfere with the rhamnolipid resonances. Titrations of each indicator solution in the absence of rhamnolipid were performed by measuring pH potentiometrically and recording the 1H NMR spectrum. The chemical shifts of the indicator resonances were used to calculate the pH of rhamnolipid solutions during the spectrochemical titration. Titration curves for the indicators are provided in Figure S2 in Supporting Information. The inset in Figure 3 shows an expanded region of the 1H NMR spectrum containing the resonances used to monitor the rhamnolipid titration. At pH 7.42, the rhamnolipid resonances of the inequivalent methylene protons (Ha and Hb) on the carbon adjacent to the carboxylate moiety occur at 2.4871 ppm for Ha and 2.4761 ppm for Hb. Plots of δobs as a function of pH for both rhamnolipid resonances are shown in Figure 4. At the lowest pH (39) Gutowsky, H. S.; Saika, A. J. Chem. Phys. 1953, 21, 1688-1694. (40) Loewenstein, A.; Roberts, J. D. J. Am. Chem. Soc. 1960, 82, 2705-2710.

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Figure 3. 1H NMR spectrum of rhamnolipid and indicator compounds at pH 7.42. Numeric labels for imidizole (I), phthalate (P), formate (F), and rhamnolipid (R) identify peaks used in the pKa determination as summarized in Table 1. Inset: expansion of region containing rhamnolipid resonances.

measured of 4.12, the chemical shifts of Ha and Hb are 2.6101 and 2.5971 ppm, respectively. Although more acidic solutions were prepared, the chemical shifts of the methylene protons could not be accurately measured for rhamnolipid solutions at pH 6. Thus, only rhamnolipid solutions above the cmc could be studied. ATR-FT-IR spectra in the region from 900 to 3000 cm-1 from 300 µM rhamnolipid solutions as a function of pH are shown in (41) Gershevitz, O.; Sukenik, C. N. J. Am. Chem. Soc. 2004, 126, 482-483. (42) Gomez-Fernandez, J. C.; Villalain, J. Chem. Phys. Lipids 1998, 96, 41-52. (43) Zscherp, C.; Schlesinger, R.; Tittor, J.; Oesterhelt, D.; Heberle, J. Proc. Nat. Acad. Sci. U.S.A. 1999, 96, 5498-5503.

Table 1. Fitting Parameters from 1H NMR Spectrochemical Titration for pH Indicators and Rhamnolipid fit parameters from 1H resonance phthalic acida

P1

P2

Ka1 Ka2 δH2A (ppm) δHA (ppm) δA- (ppm)

(1.47 ( 0.25) × 10-3 (1.05 ( 0.03) × 10-5 7.8298 ( 0.0023 7.7693 ( 0.0023 7.4810b

(1.64 ( 0.11) × 10-3 (1.03 ( 0.03) × 10-5 7.7111 ( 0.0015 7.5965 ( 0.0015 7.4154b

imidazole

I1

I2

Ka δHA(ppm) δA- (ppm)

(6.99 ( 0.16) × 10-8 8.6931b 7.7900b

(6.13 ( 0.10) × 10-8 7.4890b 7.1509b

formic acid

F

Ka δHA (ppm) δA- (ppm)

(2.92 ( 0.04) × 10-4 8.2467 ( 0.00 8.4594b

monorhamnolipid Ka δHA (ppm) δA- (ppm)

Ha (4.09 ( 0.39) × 2.6717 ( 0.0088 2.4857b

Hb 10-5

(4.00 ( 0.35) × 10-5 2.6564 ( 0.0079 2.4750b

a The phthalic acid results were fit to an equation for a diprotic acid, similar to eq 3, as described in Supporting Information. b Parameters fixed in Scientist fit of data.

Figure 4. 1H NMR chemical shift versus pH titration curves for the rhamnolipid resonances (a) Ha and (b) Hb as fit by Scientist (see Table 1 for fit parameters.)

Figure 5. In contrast to the situation for NMR spectroscopy in which a single, exchange-averaged resonance is observed whose chemical shift is defined by eq 4, the proton-exchange kinetics are slow on the time scale of the FT-IR measurement, and thus, ATR-FT-IR spectra contain distinct peaks for the acid and conjugate base forms of the rhamnolipid. The relative intensities of these peaks change as a function of pH, and this change can be used as the basis of a pKa determination. The major peaks in the spectrum of the rhamnolipid are the ν(C-H) modes between 2825 and 2950 cm-1 and the carboxylic acid stretching modes between 1500 and 1740 cm-1. The broad peak at 1735 cm-1 contains two bands due to the carbonyl ν(CdO)44 modes of the ester group and the carboxylic acid moiety, and the band at 1564 cm-1 corresponds to the νas(COO) mode of the rhamnolipid conjugate base carboxylate group.45 The νs(COO) mode of the conjugate base carboxylate at 1406 cm-1 is only visible at very high pH (data not shown). The fingerprint region below 1500 cm-1 is characterized by overlapping peaks and complicates the peak assignment to specific rhamnolipid group frequencies. The more prominent peaks are the δ(CH) modes at 1462 and 1385 cm-1, the series of bands associated (44) Max, J.; Chapados, C. J. Phys. Chem. A 2004, 108, 3324-3337. (45) Kakihana, M.; Nagumo, T.; Okamoto, M.; Kakihana, H. J. Phys. Chem. 1987, 91, 6128-6136.

Figure 5. ATR-FT-IR spectra as a function of pH for 300 µM rhamnolipid solutions at a ZnSe IRE.

with ester ν(C-O-C) vibrations between 1300 and 1100 cm-1, and the rhamnose moiety 2° alcohol ν(C-C-O)op mode at 1053 cm-1. Two significant observations can be made by comparison of the monorhamnolipid ATR-FT-IR spectra in solutions of different pH. First, as pH decreases, the overall infrared spectral intensity increases by as much as a factor of 5 between pH 10.0 and 2.5. Since intensity in ATR-FT-IR spectroscopy is directly related to concentration in the evanescent field region immediately adjacent to the IRE surface, and since the charge on the hydrophilic ZnSe Analytical Chemistry, Vol. 78, No. 22, November 15, 2006

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IRE crystal does not change in the pH range used in this study, this substantial increase in intensity implies significant structural changes of the rhamnolipid biosurfactant in the evanescent field region, probably due to changes in aggregation-state-induced adsorption, as pH decreases. The hypothesis of aggregation-state changes in the rhamnolipid is corroborated by the solution dynamic light scattering data described above in Figure 1. The observed increase in aggregate size as the pH decreases is due to greater intermolecular interactions between rhamnolipid species as their charge is reduced. Such interactions also support an increase in rhamnolipid adsorption on the ZnSe IRE46 and, hence, a corresponding increase in ATR-FT-IR intensity of rhamnolipid bands at the expense of the intensity of the water bands. This effect can be seen, for example, through the increasingly negative water δ(OH) band at 1650 cm-1 as pH decreases due to the displacement of water from the evanescent field region of the IRE. The second and perhaps more important observation about the ATR-FT-IR spectra in Figure 5 is that changes occur in relative peak intensity and shape in the ν(CdO) region as the spectrochemical titration progresses. Specifically, the intensity of the carboxylate νas(COO) band at 1550 cm-1 increases with increasing pH. In addition, however, at pH 6.5, the broad peak at 1735 cm-1 develops a noticeable shoulder on the low-frequency side at ∼1700 cm-1 that corresponds to the protonated carboxylic acid, ν(COOH).47 Since it is not possible to accurately subtract the strong water absorption band at ∼1650 cm-1 that obscures the carboxylate νas(COO) band in solutions of pH below 4.5, the absorbance of the νas(COO) mode cannot be used to determine the pKa of the rhamnolipid. Even if this band were of precisely determinable absorbance, its use as a titration indicator would only allow half of the titration curve to be determined, since it disappears for solutions below the pKa in which the protonated form dominates. This limitation is further exacerbated by the inability to use quantitatively the absolute absorbance as a measure of concentration of the two rhamnolipid acid-base forms due to pH-induced changes in rhamnolipid aggregation state that affect overall ATRFT-IR absorption intensities. To avoid these difficulties, spectra were background corrected and the complex envelope in the carboxylic acid/carboxylate region fit with three peaks at 173591740 cm-1 corresponding to the ester ν(CdO) mode, at 170091720 cm-1, the ν(COOH) mode of the protonated form, and the νas(COO) band of the deprotonated form, at 1565 cm-1. The parameters resulting from these spectral fits at all values of pH are provided in Table 2. Fit spectra in this region at representative pH values throughout the spectrochemical titration are shown in Figure 6. The integrated absorbance values from these fits were used to construct the titration curve shown in Figure 7 by plotting the integrated absorbance of the ν(COOH) band normalized to the integrated absorbance of the ν(CdO) band (A[ν(COOH)]/A[ν(CdO)]) as a function of pH. Since both deprotonation and changes in aggregation-state-induced monorhamnolipid adsorption contribute to changes in the ATR-FT-IR absorption intensities, the intensities of the carboxylic acid mode are normalized to the ester ν(CdO) mode as a reporter of total monorhamnolipid concentration in the evanescent field region.

An expression that describes the titration curve in terms of the normalized ATR-FT-IR response can be obtained by taking the natural logarithm of the Ka expression and applying mass balance with rearrangement to give

(46) Lebro´n-Paler, A.; Pemberton, J. E., to be submitted. (47) Li, H.; Tripp, C. P. Langmuir 2004, 20, 10526-10533.

The A[ν(COOH)]/A[ν(CdO)]-pH data were fit to the expression in eq 7 to determine the rhamnolipid pKa. The fit is shown in

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Table 2. Spectral Fitting Results of ATR-FT-IR Spectra in the Frequency Region between 1450 and 1850 cm-1

pH 2.5 3.0 3.6 4.1 4.5 5.0 5.5 6.0 6.5 7.0 7.4 8.0 8.4

frequency (cm-1)

fwhm (cm-1)

integrated absorbance (au cm-1)

1740.1 ( 0.4 1719.4 ( 0.5 1740.4 ( 0.1 1719.6 ( 1.2 1740.4 ( 0.1 1719.8 ( 0.4 1740.3 ( 0.1 1719.6 ( 0.5 1739.9 ( 0.2 1717.6 ( 1.1 1739.0 ( 0.2 1714.4 ( 0.5 1737.6 ( 0.5 1711.8 ( 1.9 1737.6 ( 0.4 1714.7 ( 4.8 1735.7 ( 0.1 1701.0 ( 0.0 1735.7 ( 0.4 1701.0 ( 0.0 1735.7 ( 0.0 1701.0 ( 0.0 1735.7 ( 0.1 1701.0 ( 0.0 1735.7 ( 0.0 1701.0 ( 0.0

18.3 ( 0.6 43.9 ( 0.5 18.0 ( 0.7 43.0 ( 1.0 18.5 ( 0.1 44.6 ( 0.9 18.7 ( 0.3 43.5 ( 0.8 19.9 ( 0.6 42.0 ( 1.0 21.0 ( 1.0 40.0 ( 1.0 24.0 ( 1.0 38.8 ( 0.7 22.8 ( 0.6 36.0 ( 2.0 26.9 ( 0.6 29.0 ( 3.0 26.7 ( 0.1 29.0 ( 5.0 27.0 ( 0.2 26.5 ( 0.02 26.9 ( 0.5 29.0 ( 2.0 27.1 ( 0.5 26.0 ( 1.0

0.08 ( 0.02 0.29 ( 0.07 0.08 ( 0.01 0.28 ( 0.04 0.14 ( 0.01 0.48 ( 0.02 0.19 ( 0.01 0.33 ( 0.04 0.09 ( 0.02 0.20 ( 0.03 0.08 ( 0.00 0.13 ( 0.04 0.11 ( 0.05 0.12 ( 0.05 0.06 ( 0.01 0.04 ( 0.02 0.08 ( 0.01 0.03 ( 0.01 0.06 ( 0.00 0.02 ( 0.00 0.07 ( 0.01 0.02 ( 0.01 0.07 ( 0.01 0.03 ( 0.01 0.06 ( 0.01 0.02 ( 0.01

2.303(pH - pKa) ) ln

(

CHA

[HA]

-1

[HA] 1 ) 2.303(pH-pKa) CHA 1 + exp

)

(4) (5)

[HA] for the rhamnolipid can be related to the absorbance value for the ν(COOH) band at 1700 cm-1, and the total concentration of monorhamnolipid can be related to the absorbance value for the ester ν(CdO) band at 1738 cm-1 through their effective Beer’s law expressions:

A[HA] ) [HA]deff[HA] ACHA ) CHAdeffCHA

(6)

where [HA] is the molar absorptivity of the ν(COOH) band, CHA is the molar absorptivity of the ester ν(CdO) band, and deff is the effective path length of the ATR IRE dictated by the penetration depth of the IR beam. Using these Beer’s law expressions in eq 6 gives the final sigmoidal expression used for the titration curve:

A[HA] ACHA

)

CHA/[HA] 1 + exp2.303(pH -pKa)

(7)

Figure 6. Peak fits of ν(COOH/COO-) region of ATR-FT-IR spectra from 300 µM rhamnolipid solutions of pH 2.5, 4.5, 6.5, and 8.0.

Figure 7. ATR-FT-IR spectrochemical titration curve for rhamnolipid. Points represent ratio of ATR-FT-IR integrated absorbance values; red solid line represents fit to data according to eq 7. Error bars represent one standard deviation.

Figure 7 as the solid line. The pKa determined from this fit as the inflection point of this sigmoid is 4.84 ( 0.05 with a correlation coefficient of 0.995. The pKa value determined from the ATR-FT-IR spectrochemical titration is 0.7 pKa unit below that determined with potentiometry for a 300 µM solution but 0.5 pKa unit above the value determined by 1H NMR. The difference between the ATR-FT-IR and NMR

values is not surprising in light of the different concentrations used. However, the difference between the potentiometrically determined value and that obtained from ATR-FT-IR are somewhat surprising in light of the fact that identical rhamnolipid concentrations were used. One clue to the explanation for this difference comes from the observation that the absorbance of the acid form as reported by the ν(COOH) band at 1700 cm-1 never goes to zero within the experimental pH range investigated, suggesting that the species being monitored in this experiment are those in a surface-adsorbed layer on the ZnSe IRE, some of which can never be deprotonated due to charge repulsion within the surfaceadsorbed assembly. Further evidence for the adsorption of rhamnolipid under these conditions comes from quantitative determination of the rhamnolipid surface coverage using the approach of Sperline and coworkers in which surface coverage, Γ, is related to ATR-FT-IR spectral intensity.48 For pH 3 solutions in which all rhamnolipid is protonated, this relationship for the ν(CdO) band becomes

(

)

deff A ) [HA][HA]deff + [HA] 2000 Γ N dp HA

(8)

where A is the integrated absorbance of the ν(CdO) band in au (48) Sperline, R. P.; Muraldiharan, S.; Freiser, H. Langmuir 1987, 3, 198-202.

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cm-1, N is the number of internal reflections within the ATR IRE, [HA] is the molar absorptivity of the fully protonated form calculated from the corresponding transmission spectrum (in M-1 cm-2), [HA] is the rhamnolipid concentration (in M), deff is the effective depth of penetration (in cm), dp is the depth of penetration (in cm), and Γ is in moles per square centimeter. For the ν(Cd O) band at an average frequency of 1720 cm-1, values for deff and dp are 1480 and 848 nm, respectively. For an integrated absorbance of the ν(CdO/COOH) band of 0.53 au cm-1 at pH 3, and a value of [HA] of 14 703 M-1 cm-2, a surface coverage of 8.1 × 10-10 mol/ cm2 or 4.9 × 1014 molecules/cm2 is obtained. At pH 8, the integrated absorbance of 0.10 au cm-1 corresponds to a surface coverage of 1.1 × 10-11 mol/cm2 or 6.7 × 1013 molecules/cm2. Estimating a molecular surface area of ∼1 nm2 for an average rhamnolipid species from its size, the coverage at pH 8 corresponds to approximately one monolayer of rhamnolipid while that at pH 3 corresponds to several layers. Clearly, surface adsorption occurs to an extent strongly influenced by the level of rhamnolipid aggregation in solution.15 The pH dependence of rhamnolipid adsorption is being further investigated in this laboratory and will be reported at a later date. The pH-dependent surface adsorption explains why the pKa value determined from the spectral intensities differs from that determined with potentiometry on a solution of identical solution concentration. Given that the relative concentrations of surface and solution species within the ATR sampling depth change with pH, the observation of a pKa value different from the potentiometric result at an identical solution concentration is not surprising. Obviously, care must be taken when using ATR-FT-IR spectroscopy for pKa determination of such strongly surface-active systems. CONCLUSIONS The acid dissociation constant of the biosurfactant rhamnolipid produced by P. aeruginosa bacteria was successfully determined at concentrations both below and above the cmc by potentiometry

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and two spectroscopic techniques. Potentiometric titration indicates a pKa of 4.28 ( 0.16 at concentrations below the cmc and 5.50 ( 0.06 at a concentration well above the cmc. 1H NMR titration studies at a concentration close to the cmc resulted in a pKa of 4.39 ( 0.06 while a spectrochemical titration performed using ATR-FT-IR at a concentration well above the cmc yielded a value of 4.84 ( 0.05. The pKa of 5.5 corresponds to complex aggregates both in solution and adsorbed on the glass pH electrode surface, while the higher pKa value determined with ATR-FT-IR results from the dominant contributions of surfaceadsorbed rhamnolipid to the measured spectral response. The pH dependence of such adsorption further complicates the ATR-FTIR measurements. Understanding the concentration-dependent acid dissociation behavior of these rhamnolipids is central to their use in a diversity of potential applications. This work provides the first precise measurements of the acid dissociation behavior of these complex materials. Moreover, this work clearly demonstrates the power of molecularly specific spectroscopic approaches as viable alternatives to conventional potentiometric titrimetric techniques for samples such as these biosurfactants that are inherently difficult to manipulate and that possesses undesirable adsorption properties that interfere with conventional measurements. ACKNOWLEDGMENT The authors gratefully acknowledge support of this research by the National Science Foundation (CHE-0133237 to J.E.P. and R.M.M., CHE-0317114 to J.E.P. for instrumentation, and CHE0213407 to C.K.L.). SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review May 12, 2006. Accepted August 15, 2006. AC0608826