Fluorescence and Nuclear Magnetic Resonance Spectroscopic

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Fluorescence and Nuclear Magnetic Resonance Spectroscopic Studies of the Effect of the Polymerization Concentration on the Properties of an Amino Acid-Based Polymeric Surfactant Crystal W. Harrell,† Matthew E. McCarroll,‡ Kevin F. Morris,§ Eugene J. Billiot,†,| and Isiah M. Warner*,† Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803, Department of Chemistry and Biochemistry, Southern Illinois University, Carbondale, Illinois 62901, and Department of Chemistry, Carthage College, Kenosha, Wisconsin 53140 Received May 14, 2003. In Final Form: October 7, 2003 Chiral polymeric surfactants, also known as micelle polymers, have been developed over the past decade for use as chiral selectors in the analytical separation of enantiomers. In this study, fluorescence spectroscopy and pulsed field gradient NMR (PFG-NMR) were used to determine how the concentration at which the micelles were polymerized affects their size and structure. Ten different polymerization concentrations of sodium N-undecanoyl-L-valinate (L-SUV) were investigated, ranging from slightly below the critical micelle concentration (cmc) to 50 times greater than the cmc. Analysis of fluorescence probe data indicates that significant changes in the micropolarity and microviscosity of the polymer occurred as a function of the polymerization concentration. Pulsed field gradient NMR and fluorescence quenching were also used to investigate the changes in the size of the polymers as a result of the polymerization concentration. In addition, PFG-NMR revealed information concerning the polydispersity of the micelle polymers, which is a crucial factor in understanding the chiral interactions of these species.

Introduction Polymeric surfactants (micelle polymers) are macromolecules comprised of amphiphilic moieties that are covalently linked within the hydrophobic core of the molecule. They are typically produced by polymerization of an aqueous surfactant solution, and it has been shown that polymerization is inefficient at concentrations below the critical micelle concentration (cmc) of the monomeric form of the surfactant. Therefore, polymerization is typically carried out at concentrations greater than the cmc. Herein we refer to the concentration at which the surfactants are polymerized as the polymerization concentration. To our knowledge, the effect of the polymerization concentration on the structure and performance of the polymer has not been systematically studied. Therefore, a study was performed in our laboratory to examine the effect of the polymerization concentration on the properties of an amino acid-based polymeric surfactant. Fluorescence spectroscopy and pulsed field gradient NMR (PFG-NMR) spectroscopy were used to characterize the properties of a series of polymeric amino acid surfactants produced at various polymerization concentrations. Because polymerization occurs during the formation of a conventional micelle, the structure and dynamics of the micellar system will undoubtedly affect the polymerization process. The structure of a micelle is known to be dependent on both the surfactant concentration and the * Author to whom correspondence should be addressed: e-mail [email protected]. † Louisiana State University. ‡ Southern Illinois University. § Carthage College. | Current address: Department of Physical and Life Science, Texas A&M University, Corpus Christi, Texas 78412.

properties of the surfactant monomer, as well as other contributing factors such as the temperature. Two models that have been used to describe the formation of micelles are the mass action model and the phase equilibrium model.1,2 In the mass action model, micelle formation is viewed as a series of chemical equilibria, s

2S T S2 + S T S3 T ... Sn

(1)

where S is the surfactant molecule. The process of micelle formation occurs at a very rapid rate at or above the cmc. In addition, micelles exist in a state of dynamic equilibrium.2 Equation 1 postulates a continuous distribution of monomers, dimers, trimers, and so forth. Data from the literature1,2 suggest it is conceivable for micelles to be present in the isotropic regime (below the cmc) at small concentrations. However, as the concentration of surfactant is increased above the cmc, the concentration of individual micellar units should increase in solution. In contrast, the phase equilibrium model suggests that no micelles are present below the cmc and that once the cmc is reached, any excess surfactant in solution incorporates into the micellar phase, keeping the monomer concentration constant and approximately equal to the cmc.1,2 Conventional micelles (nonpolymerized) do not always allow for the adequate discrimination of analytes. The dynamic equilibrium between the monomeric and the micellar forms of surfactant results in an aggregate with only transient stability. Furthermore, in the case of normal surfactants, the presence of a distribution of monomers, dimers, trimers, and so forth may hinder selective interactions with the analyte.3 In addition, it is often (1) Nagai, K.; Elias, H.-G. Makromol. Chem. 1987, 188, 1095. (2) Hiemenz, P. C.; Rajagopalan, R. Principles of Colloid and Surface Chemistry, 3rd ed.; Marcel Dekker: New York, 1977; pp 355-399.

10.1021/la0348362 CCC: $25.00 © 2003 American Chemical Society Published on Web 12/16/2003

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necessary to operate at concentrations well above the cmc, which can vary with temperature, ionic strength, and pH.3 These are often disadvantageous in micellar capillary electrophoresis (MCE) applications, most significantly due to joule heating, which may result in significant peak broadening.4,5 To minimize the problems associated with the use of an ionic pseudostationary phase, polymeric surfactants have been developed for use in MCE. Polymeric surfactants are fixed in size and structure by covalent bonds, rather than the usual weak forces that result in the self-assembly of surfactants.3 The use of micelle polymers not only eliminates the dynamic equilibrium and, thus, decreases polydispersity3 but also has other distinct advantages over conventional micelles, such as enhanced stability, rigidity, and tolerance to high organic modifier concentrations.4-6 It is also significant that micelle polymers possess micellar character at all concentrations, reducing the need for high surfactant concentrations to increase the concentration of micelles for effective and reproducible separations to be achieved in MCE.4 The goal of this study is to determine how the polymerization concentration of the surfactant monomer affects the properties of a polymeric surfactant. The utility of and enhanced separations achieved with polymeric surfactants have been well-established.7-11 However, there is a need to optimize such polymeric surfactant systems. It has been reported12 that polymerization of surfactants to form micelle polymers below the cmc is inefficient. In contrast, when the surfactant concentration reaches the cmc, the polymerization process is more efficient because the molecules self-associate, increasing the likelihood of polymerization.12 However, a detailed study examining the effect of the surfactant concentration on the polymerization has not been reported in the literature. To assess the affects of the polymerization concentration on the properties of the polymer, fluorescence spectroscopy was used to determine the micropolarity, microviscosity, and number of repeat units of the polymers. Diffusionordered NMR spectroscopy, a technique based on PFGNMR, was used to provide additional information about the polymer size and polydispersity. Methodology Micropolarity. The polarity of a host microenvironment is one factor that affects its interactions with guest molecules. Hence, when a polar analyte (e.g., an alcohol or carboxylic acid) is in a micellar solution, it is likely to interact primarily at the interfacial region of the micelle and will be oriented such that the polar moiety is at the interface with the nonpolar moiety penetrating into the micelle core.13 In contrast, nonpolar analytes are expected to penetrate deeply into the micellar core. Recent data, (3) Palmer, C. P. J. Chromatogr., A 1997, 780, 75-92. (4) Fendler, J. H.; Tundo, P. Acc. Chem. Res. 1984, 17, 3-8. (5) Akbay, C.; Shamsi, S. A.; Warner, I. M. Electrophoresis 1997, 18, 253-259. (6) Palmer, C. P.; Khaled, M. Y.; McNair, H. M. J. High Resolut. Chromatogr. 1992, 15, 756-762. (7) Wang, J.; Warner, I. M. Anal. Chem. 1994, 66, 3773-3776. (8) Wang, J.; Warner, I. M. J. Chromatogr. 1995, 711, 297-304. (9) Agnew-Heard, K.; Sanchez Pena, M.; Shamsi, S. A.; Warner, I. M. Anal. Chem. 1997, 69, 958-964. (10) Shamsi, S. A.; Mocossay, J.; Warner, I. M. Anal. Chem. 1997, 69, 2980-2987. (11) Dobashi, A.; Ono, T.; Hara, S.; Yamaguchi, J. Anal. Chem. 1995, 67, 3011-3017. (12) Hamid, S.; Sherrington, D. J. Chem. Soc., Chem. Commun. 1986, 936-938. (13) Laguitton-Pasquier, H.; Pansu, R.; Chauvet, J.-P.; Pernot, P.; Collet, A.; Faure, J. Langmuir 1997, 13, 1907-1917.

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however, indicates that some nonpolar molecules, such as pyrene, may not penetrate as deeply into the hydrophobic core of the micelle as was previously presumed.13 The vibronic structure of the fluorescence emission of pyrene is a good indicator of the polarity of the environment in which it is located.14-19 In the case of aqueous solutions of polymeric micelles, this is the interior of the micelle polymer. The micropolarity of pyrene solubilized by the polymeric surfactant can be used to indicate changes in the polarity of the environment surrounding the probe, which is indicative of structural or environmental changes in the polymeric micelles. Steady-State and Time-Resolved Fluorescence Anisotropy. Fluorescence anisotropy measurements were also used to characterize the micellar structure at different polymerization concentrations. Fluorescence anisotropy is defined as

r)

I| - I⊥ I| + 2I⊥

(2)

where I| and I⊥ are the fluorescence emission intensities measured with polarization, parallel and perpendicular to the polarization of the excitation radiation. When a sample is excited with plane-polarized light, the resulting emission will be partially polarized. Depolarization can occur by intrinsic and extrinsic mechanisms.20,21 Forms of extrinsic depolarization include rotational diffusion, which is typically the dominant depolarization mechanism. Molecular properties that affect the rotational motion include the size, shape, and segmental flexibility of a macromolecule.20 The effects of rotational diffusion on the fluorescence anisotropy can be described by use of the Perrin equation,

r)

r0 1 + τ/φ

(3)

where r0 is the intrinsic anisotropy observed in the absence of rotational motion, r is the measured anisotropy, τ is the fluorescence lifetime, and φ is the rotational correlation time. Probe movement within the micelle can be monitored by measuring the (steady-state) anisotropy (r) because the microviscosity of the interior influences the rotation of the probe20 as well as the overall motion of the micelle. Whether or not the motion of the larger body significantly affects the anisotropy depends on the degree of binding interaction between the probe and the micelle. If there is significant interaction and binding, the motion of the probe will be coupled to that of the micelle. Therefore, it is expected that steady-state anisotropy measurements will reflect changes in the size, shape, and interactions of macromolecules. Perylene was chosen for the study reported here as a result of its convenient lifetime (5.35 ns in cyclohexane) and its structural similarity to pyrene. Estimation of the microviscosity can lead to further insight into the structure of the solubilization site of pyrene used in the polarity (14) Nakajima, A. Bull. Chem. Soc. Jpn. 1971, 44, 3272-3277. (15) Nakajima, A. Spectrochim. Acta, Part A 1974, 30, 860-862. (16) Nakajima, A. J. Mol. Spectrosc. 1976, 61, 467-469. (17) Nakajima, A. J. Lumin. 1976, 11, 429-432. (18) Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. Soc. 1977, 99, 2039-2044. (19) Parthasarathy, R.; Labes, M. M. Langmuir 1990, 6, 542-547. (20) Lakowicz, J. Principles of Fluorescence Spectroscopy; Plenem Press: New York, 1983. (21) McCarroll, M.; Toerne, K.; von Wandruszka, K. Langmuir 1998, 14, 2965-2969.

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study.19 It should be noted that steady-state anisotropy values are altered by binding interactions.21 However, if the rotation is hindered, there is no way to discern this phenomenon from changes in microviscosity.20,21 For these reasons, microviscosity data are presented as steady-state anisotropies rather than actual viscosity values. To determine whether perylene has hindered rotation in the micelle, the time-dependent decay of the fluorescence anisotropy (dynamic anisotropy) was measured. Anisotropy measurements provide supplemental information about molecular motion and can indicate whether the fluorophore is free to rotate or whether its surrounding environment constrains its motion. In addition, anisotropy measurements can indicate whether several rotational processes or multiple environments are present and may be sensitive to the segmental motion of the polymer as well as the rotation of the entire polymer.20 The timedependent decay [r(t)] of a fluorophore can typically be represented by

r(t) ) r0

∑i fie-t/φ

i

(4)

where r0 is the anisotropy observed in the absence of rotational diffusion, t is time, ×a6i is the fractional contribution of the total fluorescence for the species i, and φi is the rotational correlation time of that species. Another consideration is the possibility of the fluorophore’s rotational motion being hindered. If the rotational motion of the fluorophore is hindered, then the anisotropy will not decay to 0. Fluorescence Quenching. Turro and Yekta23 were the first to use fluorescence quenching to determine the number of repeat units (N) of micelles. The method is based on quenching when both the fluorophore and the quencher are present in the micelle. Under such conditions, the fluorescence is completely quenched. Therefore, fluorescence is only observed from micelles that contain a probe and do not contain a quencher. Consequently, the fluorescence intensity in the presence (I) and the absence (I0) of quencher is dependent on the quencher (Q) and micelle (M) concentrations, as shown below:

I0/I ) e-[Q]/[M]

(5)

The fluorescence quenching can be related to the concentration of surfactant, [SURF], the cmc, and the number of repeat units, N, by

I0 [Q]N ln ) I [SURF] - cmc

(6)

In the case of polymeric surfactants (micelle polymers), the surfactant concentration need not be adjusted for a cmc because there are no monomers in equilibrium with the polymeric surfactant. A plot of I0/I versus [Q] gives a slope of N/([SURF] - cmc) and allows the determination of the number of repeat units.22,24 PFG-NMR. PFG-NMR is a well-established method for characterizing the overall molecular size and shape of macromolecules in solution.25-27 By use of this approach, (22) von Wandruska, R. Crit. Rev. Anal. Chem. 1992, 23, 187-215. (23) Turro, N. J.; Yekta, A. J. Am. Chem. Soc. 1978, 100, 5951-5952. (24) Rodgers, M. A. J.; Da Silvae Wheeler, M. F. Chem. Phys. Lett. 1978, 53, 165-169. (25) Stilbs, P. Prog. Nucl. Magn. Reson. Spectrosc. 1987, 19, 1-45. (26) Callaghan, P. T. Principles of Nuclear Magnetic Resonance Microscopy; Oxford University Press: New York, 1993. (27) Johnson, C. S., Jr. Prog. Nucl. Magn. Reson. Spectrosc. 1999, in press.

Figure 1. (a) Radio-frequency pulses and free induction decay and (b) magnetic field gradient pulsed for the BPLED pulse sequence. Diffusion occurs during the delay time ∆. The final delay time Te prevents phase distortions from eddy currents.

the tracer diffusion coefficient, D, of a polymer can be measured without introducing either isotopic or chemical probes into the solution. This is because the tracer diffusion coefficient is inversely proportional to the hydrodynamic radius (rh) of the polymer via the Stokes-Einstein equation. Therefore, polymers of different sizes will diffuse at different rates. All PFG-NMR experiments performed in this study were carried out using the bipolar longitudinal encode-decode (BPLED) pulse sequence shown in Figure 1.28 In the BPLED experiment, the transverse evolution time is kept at a minimum throughout the pulse sequence, and, thus, the effect of spin relaxation is governed by T1 rather than by T2. In addition, the time-varying magnetic fields used in NMR diffusion measurements are known to induce eddy currents in the metal structures of the NMR spectrometer surrounding the gradient coil.26,29 These currents have associated magnetic fields that lead to distortions of both the amplitude and the phase of the spectral components in the Fourier transformed spectra. In the BPLED experiment, the use of both bipolar gradient pulses separated by a 180° radio-frequency pulse and a longitudinal relaxation time, Te, minimizes the effects of these eddy currents. In the PFG-NMR experiments, the NMR signal is measured in the presence (I) and absence (I0) of the gradient. The delays between the bipolar pulse pair, τ, gradient pulse duration, δ, diffusion time, ∆, and eddy current delay time, Te, are all held constant while the amplitude of the gradient pulse, G, is varied. The resulting diffusion-dependent attenuation of the NMR signal, I, is given by eq 7, that is,

I ) I0 exp[-D(∆ - δ/3 - τ/2)γ2G2δ2]

(7)

where D is the diffusion coefficient, γ is the magnetogyric ratio, G and δ are the amplitude and duration of the bipolar gradient pulse, respectively, ∆ is the diffusion delay time, τ is the delay between the bipolar pulse pair, and I0 is the peak area in the absence of the gradient. A plot of the natural log of the polymer peak area versus G2 was prepared for each sample to determine the polymer diffusion coefficient. When the resulting plot was linear, it was concluded that the polymer sample was characterized by a very narrow or monodispersed distribution of diffusion coefficients. For these monodisperse samples, the diffusion coefficient of the polymer was obtained from a single-parameter least squares regression fit of the data to eq 7. (28) Wu, D.; Chen, A.; Johnson, C. S., Jr. J. Magn. Reson. 1995, 115, 260-264. (29) Gibbs, S. J.; Johnson, C. S., Jr. J. Magn. Reson. 1991, 93, 395402.

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Scheme 1. Structure of the Amino Acid-Based Surfactant Used in This Study and a Simplified Depiction of the Polymerization Process

In polymer samples that exhibit polydispersity, plots of the natural log of the peak area versus G2 show significant departure from linearity. This deviation has been shown to exist when the polydispersity of the polymer is greater than 1.5.30-32 For a polydispersed sample, data analysis methods must be employed to invert the nonexponential PFG-NMR data set into a diffusion coefficient distribution, G(D). For this purpose, we have employed the constrained regularization program Contin to calculate diffusion coefficient distributions for the polydisperse samples.33 The Contin analysis requires that high-quality PFG-NMR data be collected over a wide range of gradient amplitudes and that assumptions be made about the maximum and minimum diffusion coefficients in the sample. Even with this incorporation of a priori knowledge, the calculation of G(D) for any data set does not provide a unique solution. The Contin analysis approximates a single solution using the principle of parsimony. Therefore, of all of the solutions that fit the data, the simplest distribution function is chosen as the best solution. The smoothness of the distribution function is often an important criterion for choosing the best solution.34 The Contin analysis has been fully implemented for the analysis of PFG-NMR data sets and has been employed in the study of phospholipid vesicles,35 polymer-surfactant binding,30 humic acids,36 and polymer-molecular-weight distributions.37 (30) Morris, K. F.; Johnson, C. S., Jr.; Wong, T. C. J. Phys. Chem. 1994, 98, 603-608 (31) Raghavon, R.; Mavery, T. L.; Blum, F. D. Macromolecules 1987, 20, 814-818. (32) Callaghan, P. T.; Pinder, D. N. Macromolecules 1985, 18, 373379. (33) Provencher, S. W. Comput. Phys. Commun. 1982, 27, 213227. (34) Morris, K. F.; Johnson, C. S., Jr. J. Am. Chem. Soc. 1993, 115, 4291-4299. (35) Hinton, D. P.; Johnson, C. S., Jr. J. Phys. Chem. 1993, 97, 90649072. (36) Morris, K. F.; Dixon, A. P.; Cutak, B.; Larive, C. K. Manuscript in preparation.

Once Contin is used to resolve a diffusion distribution, an average diffusion coefficient for the polydispersed sample can be calculated by taking the ratio of the first and zeroth moments of the distribution function,30 that is, Dave ) M(1)/M(0), where

M(n) )

∫0∞DnG(D) dD

(8)

Experimental Section Polymerization of Various Sodium N-Undecanoyl-Lvalinate (L-SUV) Concentrations. Aqueous solutions of L-SUV, synthesized in our laboratory, were prepared at concentrations of 20, 40, 60, 80, 100, 200, 400, 600, 800, and 1000 mM of the monomer using water treated to an 18-MΩ resistivity. The surfactant, L-SUV, was then polymerized by 60Co γ irradiation at the various concentrations of L-SUV just mentioned (Scheme 1). The cmc of L-SUV was determined to be 21 mM by use of surface tensiometric measurements.8 The solutions were irradiated by a 60Co source for a period of 1 week and were subsequently lyophilized to yield a solid polymeric surfactant salt. Fluorescence Instrumentation. The micropolarity, steadystate fluorescence anisotropy, and fluorescence quenching measurements were performed on a SPEX model F2T211 spectrofluorometer equipped with a thermostated cell housing and a thermoelectrically cooled Hamamatsu R928 photomultiplier tube operated in the single-photon counting mode. In the case of the micropolarity and quenching studies, band passes of 4.2 and 1.7 nm were used respectively for the excitation and emission wavelengths. For the microviscosity studies, band passes of 3.4 nm were used for both excitation and emission wavelengths. In the case of pyrene polarity measurements, an excitation wavelength of 334 nm was used and the emission intensities were recorded at 372 and 383 nm. In the case of perylene, the fluorescence emission was monitored with an excitation wavelength of 414 nm and an emission wavelength of 445 nm. (37) Chen, A.; Wu, D.; Johnson, C. S., Jr. J. Am. Chem. Soc. 1993, 117, 7965-7970.

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Time-dependent anisotropy decays were measured by use of an LS 100 fluorescence lifetime system (Photon Technology International, Inc.). A nitrogen flash lamp source was used for excitation. Time-correlated single-photon counting was used to aquire the fluorescence decays, and the software package provided by Photon Technologies International was used to deconvolve the instrument response function. Both the instruments used for steady-state and anisotropy measurements were equipped with Glann-Thompson polarizers. All the anisotropy measurements were corrected using the G-factor parameter for correcting instrumental polarization bias. PFG-NMR Spectrometer. All NMR experiments on the polymerized surfactant samples were performed by use of a Bru¨ker DPX-300 spectrometer equipped with a 5-mm actively shielded z-gradient probe. The maximum gradient strength of 50.3 G/cm was determined by performing a pulsed gradient NMR experiment on β-cyclodextrin in D2O (D ) 3.23 × 10-10 m2 s-1).43 The intensity and duration of the magnetic field gradient pulses were controlled by the spectrometer’s Silicon Graphics O2 workstation. Data Acquisition. All diffusion experiments were performed at 298 K using the BPLED pulse sequence (vide supra).28 In all the diffusion studies, ∆, τ, and δ were respectively set at 250, 1.2, and 2.0 ms. For the polymeric surfactant samples with polymerization concentrations ranging from 20 to 100 mM, 14 free induction decays were collected in each diffusion experiment, with gradient strengths, G, ranging from 2.5 to 32.7 G/cm. For the samples polymerized at concentrations ranging from 200 to 1000 mM, 27 free induction decays were collected with gradients in the range of 2.5 to 37.8 G/cm. Each free induction decay was acquired with a spectral width of 6173 Hz and 8000 data points. Data Processing and Calculations of Diffusion Coefficients. After data acquisition, the free induction decays collected for each experiment were apodized with 5-Hz line broadening, Fourier transformed, and phased, and the spectral region from approximately 1.0 to 2.5 ppm was integrated. For the polymeric surfactant samples with polymerization concentrations of 20-100 mM, diffusion coefficients were calculated from the slope of the line obtained by plotting ln I versus [∆ (δ/3) - (τ/2)]γ2G2δ2 using the software package Sigma-plot. In the samples polymerized at concentrations ranging from 200 to 1000 mM, plots of ln I versus [∆ - (δ/3) - (τ/2)]γ2G2δ2 were also prepared. These plots, however, were nonlinear, indicating that the samples were polydispersed. Therefore, the constrained regularization program Contin33 was used to calculate diffusion coefficient distributions for these samples. The use of Contin to resolve diffusion distributions from pulsed gradient NMR data has been described in detail elsewhere.30,34,37 The resulting 50point diffusion coefficient distributions calculated by Contin were then plotted by use of the software package Sigma-plot. An average diffusion rate was calculated by dividing the first and zeroth moments of the distribution, that is,

∑G D i

〈D〉 )

i

i

∑G

(9) i

i

Fluorescence Solutions. A Stock solution of pyrene was prepared in cyclohexane. Aliquots were then transferred into 10 separate vials, and the cyclohexane was evaporated. Aqueous polymer solutions were then added to each of the vials, sonicated for 5 min, and allowed to equilibrate prior to measurement. Unless otherwise noted, all polymer concentrations stated are the (38) Morio, Y. Micelles, Theoretical and Applied Aspects; Plenum Press: New York, 1992; Chapter 4. (39) Anton, P.; Ko¨berle, P.; Laschewsky, A. Makromol. Chem. 1993, 194, 1-27. (40) Laschewsky, A. Adv. Polym. Sci. 1995, 124, 3-85. (41) Strauss, U. P.; Barbieri, B. W. Macromolecules 1982, 15, 13471349. (42) Barbieri, B. W.; Strauss, U. P. Macromolecules 1985, 18, 411414. (43) Zdanowicz, V. S.; Strauss, U. P. Macromolecules 1993, 26, 47704773.

Figure 2. Variation of steady-state anisotropy (r) and the I/III ratio (372/383 nm) of pyrene vibronic bands (polarity) with the polymerization concentration. equivalent monomer concentration (EMC). In the case of the anisotropy experiment, the perylene and polymer concentrations were 1.00 × 10-5 and 1.67 × 10-4 M, respectively. In the polarity study, the pyrene and polymer concentrations were 1.00 × 10-6 and 4.00 × 10-4 M, respectively. In samples containing perylene, a similar procedure was followed; however, chloroform was used instead of cyclohexane. For the quenching study, two solutions were prepared, both of which contained 50 mM polymer and 1.00 × 10-4 M pyrene. One of the solutions, however, contained the quencher, cetylpyridinium chloride, at a concentration of 1.5 × 10-3 M. Therefore, when the aliquots of the solution containing quencher are incrementally added to the other solution, the surfactant and probe concentrations remain constant and the quencher concentration increases. After each aliquot of quencher was added and allowed to equilibrate, the fluorescence intensity was measured and recorded.

Results Steady-State Fluorescence and Polarity Measurements. To better understand the microenvironmental properties of the polymeric surfactants, micropolarity and microviscosity were examined. The fluorescence anisotropy of the probe perylene was used to evaluate changes in the microviscosity of the solubilization site within the polymer, recognizing that a higher anisotropy is indicative of a more viscous environment. Figure 2 shows the steadystate anisotropy of perylene and the pyrene I/III as functions of the polymerization concentration. The anisotropy was found to vary significantly, and a minimum was observed at a polymerization concentration of approximately 200 mM, indicating that the probe was localized in a less viscous environment. While the probe anisotropy reached a minimum at a polymerization concentration of 200 mM, the micropolarity (pyrene I/III) increased as a function of the polymerization concentration, reaching a maximum at 200 mM. These data indicate that the polymers produced at 200 mM possess solubilization sites that are less viscous and more polar. It should be noted that these observations could result from changes in the structure of the polymer or changes in the solubilization site of the probes. In either case, it is clear that the type of interaction experienced by the probe is strongly dependent on the concentration of the surfactant during polymerization. Differences in the polymeric surfactant interior, such as the size, shape, and viscosity, affect the polarity of the environment surrounding the probe. The changes in the molecular properties listed above may occur from decreased packing or an expansion (becoming more linear) of the polymer. At polymerization concentrations greater than 400 mM, the polymer packing appears to be altered, possibly as a result of changes in shape to a more open

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Figure 3. Anisotropy at t ) 0 (r′) observed from dynamic anisotropy measurements as a function of the polymerization concentration.

Figure 4. Variation of the number of repeat units ()) and normalized diffusion coefficients (0) versus the polymerization concentration.

Table 1. Presentation of the Size Determination Data Using Fluorescence Quenching and PFG-NMRa polymerization concentration (mM)

MWb (D)

Nb

Nc

20 40 60 80 100 200 400 600 800 1000

8908.5 ( 1.2 14 280.0 ( 1.8 9418.1 ( 1.1 9945.5 ( 1.3 7728.0 ( 1.5 12 218.3 ( 2.3 e e e e

29 47 31 33 25 40 e e e e

12 ( 1.3 30 ( 0.8 30 ( 0.8 30 ( 1.0 28 ( 2.3 34 ( 2.7 52 ( 2.2 78 ( 2.8 75 ( 4.5 83 ( 4.5

diffusion coefficientd dis(cm2 s-1) persityd 1.03 × 10-6 1.07 × 10-6 9.18 × 10-7 9.19 × 10-7 9.92 × 10-7 1.10 × 10-6 1.21 × 10-6 1.20 × 10-6 1.32 × 10-6 2.74 × 10-6

mono mono mono mono mono poly poly poly poly poly

aThe errors in the diffusion coefficients are expected to be less than 5% and approximately 10% for the monodisperse and polydisperse samples, respectively. b Determined by analytical ultracentrifuge sedimentation equilibrium experiments. c Determined by fluorescence quenching experiments. d Determined by diffusion order NMR experiments; mono ) monodisperse, and poly ) polydisperse. e Data not obtained using this technique.

structure. This speculation is supported by the changes observed in the number of repeat units of the polymers (vide infra). Time-Dependent Measurements. Time-dependent anisotropy measurements provide additional information about the polymers. The anisotropy at time 0 is typically equal to the intrinsic anisotropy, r0 (0.36).20 In this system, however, the anisotropy measured at time 0 was lower in value than the intrinsic anisotropy. This can be explained by the rotation of the probe at a rate faster than that which could be resolved by our instrumentation. Therefore, we refer to the anisotropy measured at t ) 0 as an apparent intrinsic anisotropy (r′). Figure 3 shows that, as the polymerization concentration increases from 20 to 400 mM, the apparent intrinsic anisotropy (r′) decreased. This decrease in r′ likely represents changes in a component of the rotation with a rapid anisotropy decay that cannot be resolved (φ < ns). However, it is not clear whether the decrease in r′ is due to changes in the rotational correlation time or a change in the relative population (fi) of the fast component. The anisotropy decays (data not shown) revealed that all of the polymerization concentrations have a limiting anisotropy (r∞), suggesting that the probe rotation is hindered or bound to a certain degree by the polymeric surfactant. The decrease in r′ is likely due to less hindered rotation, which correlates well with the steady-state microviscosity data (vide supra). Size Determination Using Fluorescence Quenching and PFG-NMR. The number of repeat units and diffusion coefficients obtained from fluorescence quenching

Figure 5. Decay of the NMR peak area with the gradient strength for polymerization concentrations of 100, 200, and 800 mM. A nonlinear decay is observed for the 200 and 800 mM samples, indicating that those samples exhibit polydispersity.

and PFG-NMR are shown in Table 1. The number of repeat units obtained using the fluorescence quenching technique ranged from 13 to 83. With the exception of the polymer produced at a polymerization concentration of 20 mM (cmc ) 21 mM), the number of repeat units remained relatively constant up to a polymerization concentration of 200 mM. At concentrations above 200, the number of repeat units increased (Figure 4). Additional information pertaining to the size of the polymers was obtained from PFG-NMR (Table 1). The diffusion coefficients of the polymers produced at polymerization concentrations ranging from 20 to 200 mM did not vary substantially. These findings are in agreement with the fluorescence quenching data at lower polymerization concentrations, where the diffusion and number of repeat units vary inversely. At concentrations greater than 200 mM, the inverse relationship does not hold well because both the diffusion constants and the number of repeat units increase, albeit at different rates. While we do not have a completely satisfying explanation for this apparent anomaly, we suspect that polydispersity and the limited sensitivity of NMR plays a significant role. The NMR experiment is not sensitive to species at concentrations less than ∼10-3 M, whereas the fluorescence quenching experiment is expected to have a much higher sensitivity. Because both techniques measure an average value, it is reasonable to expect that the values could be skewed in polydispersed samples.

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fluorescence and separation studies and, therefore, intermolecular aggregation should not be a factor in these experiments. Discussion

Figure 6. Diffusion coefficient distribution function calculated with Contin for the polymeric surfactant sample polymerized at 800 mM.

Figure 7. Change of the diffusion coefficient (D) with the variation of the EMC of poly(L-SUV) polymerized at 100 mM. The sudden drop in D represents the beginning of intermolecular aggregation.

Fortunately, the PFG-NMR experiment also allows for an estimation of the polydispersity, as evidenced by nonlinear plots of ln I versus [∆ - (δ/3) - (τ/2)]γ2G2δ2. Linear plots were observed for polymerization concentrations up to 100 mM (Figure 5), signifying monodispersed polymeric surfactants. Nonlinear plots were obtained for polymers with polymerization concentrations greater than 200 mM. An even greater polydispersity was observed at polymerization concentrations greater than 400 mM. A typical distribution function [G(D)] from the Contin analysis is shown in Figure 6, which is essentially a massweighted distribution diffusion coefficient. Another characteristic investigated using PFG-NMR was intermolecular aggregation of the polymeric surfactants after polymerization. The polymer produced at a polymerization concentration of 100 mM was examined by measuring the diffusion coefficients at various polymer concentrations (shown as the EMC). A plot of the diffusion coefficient as a function of the polymer concentration is shown in Figure 7. At the lower concentrations, the diffusion coefficients are similar, suggesting no intermolecular aggregation. At concentrations greater than 15 mM, however, the diffusion coefficients decrease significantly, presumably as a result of intermolecular aggregation. It should be noted that polymer concentrations of less than 15 mM were used in the

From the trends observed in the polarity and anisotropy data, it appears that structural changes occurred at polymerization concentrations in the range of 200-400 mM. This is further supported by the changes observed in the number of repeat units and PFG-NMR diffusion coefficients. Interestingly, the concentration at which the polarity and microviscosity measurements reached maximum and minimum values corresponds to the onset of the polydispersity, as evidenced by the PFG-NMR experiments. Nagai and Elias1 have reported that the chemical microstructure and the overall structure of the monomeric micelle are not retained during or after polymerization. However, the demonstrated dependence of the polymer properties on the polymerization concentration indicates that the micelle structure significantly affects the polymerization. Because substantial changes in the polymer size were not observed from the polymerization concentrations ranging from 40 to 200 mM, the observed trends are likely a result of changes in the polymer structure rather than in the polymer size. At higher polymerization concentrations, changes in the shape and size may play a more significant role. It is well-known that conventional micelles may undergo significant changes in micellar structure as a result of changes in the surfactant concentration. It is expected that the polymeric micelles produced at various concentrations will resemble conventional micelles observed at commensurate surfactant concentrations. Therefore, the larger number of repeat units that are observed at polymerization concentrations above 200 mM may indicate a shape transition, such as that from a sphericalto a rod-shaped micelle, as is often observed in conventional surfactant solutions.38 Specifically concerning polymeric micelles, three models have been proposed, the polymers of which vary in structure and characteristics.39,40 The first model was proposed from the work of Strauss et al.41-43 and is described as a “local micelle”. In this structure, it is presumed that the aggregation of a limited number of surfactant units occurs. This model is not dependent on the degree of polymerization and allows for aggregation of the macromolecules. Efficient aggregation of the macromolecules depends on the flexibility of the polymer backbone. This model tends to describe more hydrophobic polymers. The “molecular micelle” model has evolved from the studies of Chu and Thomas44,45 and Elias et al.46-50 This model is described as the intramolecular aggregation of polymeric surfactant chains into one macromolecule model, where the degree of polymerization is equivalent to the number of repeat units. Molecular micelles have less flexible backbones, and the hydrophobic tails are more shielded from water.39,40 (44) Chu, D. Y.; Thomas, J. K. Macromolecules 1987, 20, 2133-2138. (45) Chu, D. Y.; Thomas, J. K. Macromolecules 1991, 24, 2212-2216. (46) Watterson, J. G.; Elias, H. G. Kolloid Z. Z. Polym. 1971, 249, 1136-1143. (47) Laesser, H. R.; Elias, H. G. Kolloid Z. Z. Polym. 1972, 250, 4657. (48) Laesser, H. R.; Elias, H. G. Kolloid Z. Z. Polym. 1972, 250, 5863. (49) Laesser, H. R.; Elias, H. G. Kolloid Z. Z. Polym. 1972, 250, 6475. (50) Laesser, H. R.; Elias, H. G. Kolloid Z. Z. Polym. 1972, 250, 344351.

Amino Acid-Based Polymeric Surfactant

The “regional micelle” model is a model intermediate to the previous models.39,40,51 The aggregation of the surfactant monomers is affected by the aggregation of the individual segments of the polymer. This model is versatile and can account for a gradual transition from intra- to intermolecular aggregation.40 To date, there is, however, no comprehensive theory of polymeric micelles.40 The polymers described in this study are best described by a regional micelle model. While the exact structure and dynamics cannot be determined with absolute certainty, the data presented in this study provide important evidence leading toward a better understanding of this type of system. The polymers exhibit segmental motion (flexibility), changes in micropolarity and microviscosity, and intermolecular aggregation. More importantly, these parameters are significantly affected by the concentration at which the surfactants are polymerized. Conclusions It is clear that the polymerization concentration of polymeric surfactants has a significant effect on the (51) Morishima, Y.; Tsuji, M.; Kamachi, M.; Hatada, K. Macromolecules 1992, 25, 4406-4410.

Langmuir, Vol. 19, No. 26, 2003 10691

structure and interactions of these macromolecules. Examination of data from fluorescence probe experiments and PFG-NMR measurements indicate the onset of structural diversity and polydispersity at polymerization concentrations in the 200-400 mM range. While a precise description of the structural changes remains elusive at this time, it is clear that these effects should be considered when producing polymeric surfactants. Furthermore, it has been demonstrated that intermolecular aggregation of the polymers occurs at relatively low concentrations (∼15 mM) and is a factor that should be considered. Further studies are necessary before a conclusive understanding of the structure and dynamics of micelle polymers is at hand. Acknowledgment. This work was supported by grants from the National Science Foundation (Grant CHE9632916) and the National Institute of Health (Grant R1GM39844D). Isiah M. Warner also acknowledges the Philip W. West endowment for partial support of this research. Kevin F. Morris acknowledges the Camille Dreyfus Foundation for support. The authors also thank Judson L. Haynes III for useful discussion. LA0348362