Oxygen Diffusion in Cross-Linked, Ethanol-Swollen Poly(vinyl alcohol

Dec 22, 2008 - and Food Safety, UniVersity of Chester, Chester, United Kingdom ... Center for Oxygen Microscopy and Imaging, University of Aarhus...
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Langmuir 2009, 25, 1148-1153

Oxygen Diffusion in Cross-Linked, Ethanol-Swollen Poly(vinyl alcohol) Gels: Counter-Intuitive Results Reflect Microscopic Heterogeneities Nickolass Bitsch Schack,†,‡ Cristiano L. P. Oliveira,‡,§ Niall W. G. Young,|,⊥ Jan Skov Pedersen,‡,§ and Peter R. Ogilby*,†,‡,§ Center for Oxygen Microscopy and Imaging, Department of Chemistry, and The Interdisciplinary Nanoscience Center (iNANO), UniVersity of Aarhus, DK-8000 Århus, Denmark, Danisco A/S, DK-8220 Brabrand, Denmark, and The Faculty of Applied and Health Sciences, EnVironmental Quality and Food Safety, UniVersity of Chester, Chester, United Kingdom ReceiVed September 15, 2008. ReVised Manuscript ReceiVed October 29, 2008 Oxygen diffusion coefficients have been determined in ethanol-swollen poly(vinyl alcohol), PVA, gels using a technique wherein oxygen sorption is optically monitored using singlet oxygen phosphorescence. Data were recorded as a function of the extent to which the PVA chains are chemically cross-linked using glutaraldehyde. Contrary to conventional expectation, the diffusion coefficients obtained increase with an increase in the extent of cross-linking. This observation is interpreted in terms of a cross-link-dependent increase in the microscopic heterogeneity of the polymer wherein dense cross-linked domains coexist with more fluid domains. It is expected that, in the latter domains, segmental motions of the macromolecule that facilitate oxygen diffusion are more readily achieved. This model of cross-link-dependent heterogeneity is supported by the results of small-angle X-ray scattering experiments. Among other things, the data reported herein provide an informative foundation for studies of small molecule diffusion in biological cells, particularly during photoinduced cell death where the hydrogel-like nature of the cell can change due to cross-linking reactions.

Introduction The diffusion of small molecules, including oxygen, in waterswollen gels has been extensively studied from a number of perspectives.1-9 One clear motivation for such work has been the use of these gels as membranes and materials in biomedical applications.10 Tissue engineering,11 drug delivery,10 and sensor coatings12,13 are examples often cited in this regard. A common and important variable in these studies is the extent to which the parent macromolecule is cross-linked.1-4 The results of these studies generally indicate that, as the given material becomes more cross-linked, the diffusion coefficient of the translating solute decreases.1-4 This is an intuitively reasonable * To whom correspondence should be addressed. E-mail:progilby@ chem.au.dk. † Center for Oxygen Microscopy and Imaging, University of Aarhus. ‡ Department of Chemistry, University of Aarhus. § The Interdisciplinary Nanoscience Center (iNANO), University of Aarhus. | Danisco A/S. ⊥ University of Chester.

(1) Korsmeyer, R. W.; Peppas, N. A. J. Membr. Sci. 1981, 9, 211–227. (2) Matsuyama, H.; Teramoto, M.; Urano, H. J. Membr. Sci. 1997, 126, 151– 160. (3) van Stroe-Biezen, S. A. M.; Everaerts, F. M.; Janssen, L. J. J.; Tacken, R. A. Anal. Chim. Acta 1993, 273, 553–560. (4) Kubin, M.; Spacek, P. Collect. Czech. Chem. Commun. 1965, 30, 3294– 3302. (5) Higuchi, A.; Fushimi, H.; Iijima, T. J. Membr. Sci. 1985, 25, 171–180. (6) Manetti, C.; Casciani, L.; Pescosolido, N. Polymer 2002, 43, 87–92. (7) Parker, J. W.; Cox, M. E. J. Polym. Sci., Part A: Polym. Chem. 1988, 26, 1179–1188. (8) Simon-Lukasik, K. V.; Ludescher, R. D. Food Hydrocolloids 2004, 18, 621–630. (9) Compan˜, V.; Tiemblo, P.; Garcia, F.; Garcia, J. M.; Guzman, J.; Riande, E. Biomaterials 2005, 26, 3783–3791. (10) Peppas, N. A.; Hilt, J. Z.; Khademhosseini, A.; Langer, R. AdV. Mater. 2006, 18, 1345–1360. (11) Drury, J. L.; Mooney, D. J. Biomaterials 2003, 24, 4337–4351. (12) Richter, A.; Paschew, G.; Klatt, S.; Lienig, J.; Arndt, K.-F.; Adler, H.-J. P. Sensors 2008, 8, 561–581. (13) Wolfbeis, O. S.; Oehme, I.; Papkovskaya, N.; Klimant, I. Biosens. Bioelectron. 2000, 15, 69–76.

observation; the density of the gel increases with an increase in cross-links which adversely influences solute translational motion. It is inferred that the latter principally reflects a decrease in the local free volume and the extent of polymer segmental motion, both of which are key factors in determining the magnitude of the diffusion coefficient.14,15 Molecular oxygen is an important solute in a plethora of systems.16,17 A study of the factors that influence the translational motion of oxygen is pertinent, in the least, in the design and construction of barrier membranes.15,18 However, as a key biological species that plays a number of roles, understanding oxygen diffusion through gel-like materials can have many farreaching ramifications. This is pertinent when one considers that a viable cell, with its protein-based cytoskeletal network, can be considered as a viscous gel.19,20 To our knowledge, studies of oxygen diffusion in gels as a function of the cross-linking extent are surprisingly limited. Nevertheless, data from two different hydrogel systems indicate that, as expected, the oxygen diffusion coefficient decreases as the extent of chemical cross-linking increases.3,4 On the other hand, with reference to experiments performed on gelatin films, it has been inferred that an observed increase in the oxygen (14) Neogi, P., Ed. Diffusion in Polymers; Marcel Dekker, Inc.: New York, 1996. (15) Paul, D. R., Yampol’skii, Y. P., Eds. Polymeric Gas Separation Membranes; CRC Press: Boca Raton, FL, 1994. (16) Lane, N. Oxygen: The molecule that made the world.; Oxford University Press: Oxford, 2002. (17) Sawyer, D. T. Oxygen Chemistry; Oxford University Press: New York, 1991. (18) Koros, W. J., Ed. Barrier Polymers and Structures; ACS Symposium Series; American Chemical Society: Washington, D.C., 1990; Vol. 423. (19) Breitenbach, T.; Kuimova, M. K.; Gbur, P.; Hatz, S.; Schack, N. B.; Pedersen, B. W.; Lambert, J. D. C.; Poulsen, L.; Ogilby, P. R. Photochem. Photobiol. Sci., published online Sept 23, http://dx.doi.org/10.1039/B809049A. (20) Hatz, S.; Poulsen, L.; Ogilby, P. R. Photochem. Photobiol. 2008, 84, 1284–1290.

10.1021/la803024h CCC: $40.75  2009 American Chemical Society Published on Web 12/22/2008

Oxygen Diffusion in Ethanol-Swollen PVA Gels

diffusion coefficient may, in fact, correlate with an increase in the extent of physical cross-linking in the gel.21 For the current study, we wanted to further investigate the extent to which cross-linking influences oxygen diffusion in solvent-swollen gels. To this end, we set out to reconfigure an experimental apparatus that we previously developed for the study of oxygen diffusion in glassy polymer films22-26 such that it could be used for solvent-swollen gels. In our technique, oxygen sorption into a given material is monitored, in time, using the phosphorescence of singlet molecular oxygen [O2(a1∆g) f O2(X3Σg-)] as a probe. In this paper, we present results obtained using ethanolswollen samples of glutaraldehyde-cross-linked poly(vinyl alcohol), PVA. Glutaraldehyde-cross-linked PVA is a commonly used material1-3 and, as such, facilitates comparisons with other published studies. The data we report herein provide an example of “contrary” behavior; the oxygen diffusion coefficient in the PVA gel increases with an increase in the extent of cross-linking.

Results and Discussion 1. Quantifying Oxygen Diffusion in Solvent-Swollen Gels. 1.1. General Approach. The intent in this study was to quantify oxygen diffusion coefficients using an approach we developed and have exploited in a number of experiments on glassy polymeric systems.22-26 In this approach, oxygen sorption into the sample under study is monitored using the 1270 nm phosphorescence of singlet molecular oxygen as the probe. Singlet oxygen is most conveniently produced by adding a small amount of a photosensitizer to the sample ( G′′, as expected for a solidlike material.35,36 Nevertheless, the data also clearly indicate that the viscoelastic response of our PVA samples depends on (1) the solvent used to swell the gel and (2) the cross-link degree. For lightly cross-linked samples (i.e., cross-link degree less than ∼0.06), both the storage and loss moduli are much greater for the ethanol-swollen samples than for the water-swollen samples. (28) Frederiksen, P. K.; McIlroy, S. P.; Nielsen, C. B.; Nikolajsen, L.; Skovsen, E.; Jørgensen, M.; Mikkelsen, K. V.; Ogilby, P. R. J. Am. Chem. Soc. 2005, 127, 255–269. (29) Pauly, S. In Polymer Handbook, 4th ed; Brandrup, J., Immergut, E. H., Grulke, E. A., Eds.; John Wiley and Sons: Hoboken, NJ, 1999; Vol. 2, pp 543-569. (30) Ware, W. R. J. Phys. Chem. 1962, 66, 455–458. (31) Sada, E.; Kito, S.; Oda, T.; Ito, Y. Chem. Eng. J. 1975, 10, 155–159. (32) Akgerman, A.; Gainer, J. L. Ind. Eng. Chem. Fundam. 1972, 11, 373– 379. (33) Bird, R. B.; Stewart, W. E.; Lightfoot, E. N. Transport Phenomena; 2nd ed.; John Wiley and Sons: New York, 2002. (34) Munk, P. Introduction to Macromolecular Science; John Wiley and Sons: New York, 1989. (35) Almdal, K.; Dyre, J.; Hvidt, S.; Kramer, O. Polym. Gels Networks 1993, 1, 5–17. (36) Terech, P.; Weiss, R. G. Chem. ReV. 1997, 97, 3133–3159.

Figure 3. Data obtained from a SAXS experiment where the log of the intensity of scattered radiation, I, is plotted against the log of the scattering vector, q. Superimposed on the data is the result of a fit to the data using eq 2. Data are from a sample with a cross-link degree of 0.067.

This is consistent with the observation that, for these lightly cross-linked materials, the samples appreciably shrink in size when water is replaced by ethanol as the swelling medium (see Experimental Section). In short, ethanol does not appear to be as “good” a solvent as water for these PVA gels, an observation which is consistent with literature reports.37 For samples with a cross-link degree greater than ∼0.06, the viscoelastic response of the ethanol-swollen gels is effectively the same as that of the water-swollen gels. In this regime, the response of the gel appears to be dominated by the cross-links. 2.2. Small-Angle X-Ray Scattering (SAXS). Upon visual inspection, our ethanol-swollen samples had a slightly cloudy appearance. This is indicative of light scattering by inhomogeneities in the sample that have a typical length scale of ∼500-1000 Å. The potential presence of such inhomogeneous domains would be quite important when interpreting our oxygen diffusion data. As such, we set out to perform a more quantitative assessment of gel homogeneity using SAXS, a technique frequently used to study gels.36,38-40 A typical data set obtained in our experiments is shown in Figure 3 where we plot the log of the intensity of scattered radiation against the log of the scattering vector, q. The intensity levels off at low values of q in agreement with typical Guinier behavior, indicating that there are density fluctuations on a comparatively large length scale.39 At high q, the scattering originates from the polymer strands.39 At intermediate values of q, there is a discrete “bump” in the profile suggesting the presence of domains in the gel that have a characteristic size. Data such as those in Figure 3 can be analyzed using a model based partly on our own work39,41 and partly on work by Geissler et al.38 In this model, we represent the intensity of scattered radiation as the sum of three terms that account for scattering by domains of three different characteristic sizes (eq 2). The term labeled “large” accounts for structures that appear at low q. The term labeled “Lorentz” describes scattering at high q (i.e., the polymer strands). The final term, labeled “domain”, is intended (37) Mu¨ller-Plathe, F.; van Gunsteren, W. F. Polymer 1997, 38, 2259–2268. (38) Geissler, E.; Hecht, A. M.; Rochas, C.; Horkay, F.; Basser, P. J. Macromol. Symp. 2005, 227, 27–37. (39) Falca˜o, A. N.; Pedersen, J. S.; Mortensen, K. Macromolecules 1993, 26, 5350–5364. (40) Falca˜o, A. N.; Pedersen, J. S.; Mortensen, K.; Boue, F. Macromolecules 1996, 29, 809–818. (41) Bunger, M. H.; Foss, M.; Erlacher, K.; Hovgaard, M. B.; Chevallier, J.; Langdahl, B.; Bunger, C.; Birkedal, H.; Besenbacher, F.; Pedersen, J. S. Bone 2006, 39, 530–541.

Oxygen Diffusion in Ethanol-Swollen PVA Gels

Figure 4. Plot of the log of the scale factors, S, from eq 2 against the cross-linking degree of our ethanol-swollen PVA gels: large structure scattering (b), domain scattering (O), and Lorentzian term (0). The lines are simply guides for the eye.

to describe inhomogeneities that occur at intermediate length scales. The “background” term refers to residual scattering by the solvent.

I (q) ) Slarge

(

1 2q Rg,large2 1+ 3Rlarge 2

)

Rlarge⁄2

1

(

+

)

+ 2q Rg,domain2 Rdomain⁄2 1+ 3Rdomain 1 + background (2) SLorentz q2Rg,Lorentz2 1+ 3

Sdomain

2

(

)

Examples of how each of these respective terms contributes to the description of our data are provided in the Supporting Information. In Figure 3, we provide an example of a fit of eq 2 to an experimental data set obtained from one of our gels. The “large”, “domain”, and “Lorentz” terms in eq 2 all have a Guinier behavior at low q and cross over to a power-law behavior at higher q. In general, we find that the exponent for the “large” structure term, Rlarge, is independent of the cross-linking degree and has a magnitude which ensures that this term falls off very quickly with increasing q. The exponent for the “domain” term, Rdomain, first increases and then decreases as the degree of crosslinking is increased. At cross-linking degrees greater than ∼0.05, Rdomain remains constant (see the Supporting Information). A plot of the respective scale factors for each of the normalized terms in eq 2 (e.g., Slarge) against the cross-linking degree of our gels is shown in Figure 4. The scale factor for the “large” structure term decreases significantly with initial increases in the crosslink density. The scale factor for the “domain” term likewise decreases significantly with an initial increase in the cross-link degree. Thereafter, there is an increase in the magnitude of this term with further increases in the degree of cross-linking. The scale factor for the “Lorentz” term effectively remains constant which is expected given the approach we use to normalize the intensity of our data (see Experimental Section). Finally, in Figure 5, we show plots of the characteristic length scales, expressed as the radius of gyration, Rg, obtained for both the “large” and “domain” terms against the cross-linking degree of our gels. Unfortunately, we do not have enough information to accurately determine Rg for the “Lorentz” term. As such, the

Langmuir, Vol. 25, No. 2, 2009 1151

Figure 5. Plot of the radius of gyration, Rg, obtained for both the “large” (b) and “domain” (O) terms against the cross-linking degree of our ethanol-swollen gels. The lines are simply guides for the eye.

corresponding radius of gyration was fixed at 10 Å, which is appropriate for scattering from polymer strands.39 The data are clearly consistent with the presence in our gels of reasonably large structural domains (i.e., Rg ∼ 20-120 Å). Most importantly, the size of these structures collectively quantified by the “domain” term in eq 2 ultimately increases with an increase in the crosslinking degree. At the limit of a cross-linking degree of 0.1, Rg for the “domain” structures is approximately the same as that for the “large” structures. In conclusion, the SAXS data recorded from our ethanolswollen gels suggest that, as the extent of cross-linking is increased up to a cross-linking degree of ∼0.05, our samples may, in fact, become somewhat more homogeneous as reflected in the decrease of the scale factors and characteristic length scales, Rg, for both the “large” and “domain” contributions.39 Nevertheless, in samples with such a low degree of cross-linking, this apparent change in homogeneity is clearly not manifested in our oxygen diffusion coefficients (Table 1). As the cross-linking degree is increased above ∼0.05, however, a clear change is seen in the SAXS data which is consistent with the fact that we also observe corresponding cross-link-dependent changes in our rheology data (Table 2) and in our diffusion coefficients (Table 1). The SAXS data suggest that, on a length scale of ∼20-120 Å, the heterogeneity of our gels in these more highly cross-linked samples increases with an increase in the cross-link density as reflected in the corresponding increases in the scale factors and Rg. As seen in Figures 4 and 5, the latter are more pronounced for the “domain” contribution. The length scale of 20–120 Å is particularly pertinent with respect to events that facilitate and/or inhibit the translational motion of a small molecule such as oxygen.25 3. The Counter-Intuitive Cross-Link-Dependent Increase in D On the basis of the most straightforward model that an increase in the extent of cross-linking in a gel will decrease both macromolecular segmental motion and polymer free volume, it is reasonable to expect that diffusion coefficients for a solute such as oxygen should decrease with an increase in the extent of cross-linking. This expectation is indeed met in the limited number of studies that have been published.3,4 In contrast, the oxygen diffusion coefficients we obtain for ethanol-swollen PVA gels increase with an increase in the extent of cross-linking (Table 1). Our data can be interpreted in terms of a cross-link-dependent increase in the microscopic heterogeneity of the polymer wherein dense cross-linked domains coexist with more fluid domains characterized by fewer cross-links. It is expected that the latter

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would be associated with a greater extent of macromolecular segmental motion that facilitates oxygen diffusion. Our independent SAXS experiments support this model of a cross-linkdependent increase in the heterogeneity of our gels. Moreover, the SAXS data suggest that this heterogeneity indeed occurs on length scales that are significant with respect to events that can influence oxygen diffusion. Our observations and model become more reasonable when it is realized that, in comparison to water, ethanol is not a “good” solvent for PVA. Although the glutaraldehyde cross-links were introduced in an aqueous solution of PVA, there may nevertheless still be an inhomogeneous distribution of these cross-links in the polymer; that is, it is reasonable to consider that, after the incorporation of one cross-link, subsequent cross-links will occur near the first where macromolecular segments may now be more judiciously positioned for reaction. When the degree of crosslinking becomes sufficiently large, these inhomogeneities in the cross-link distribution, and their effects, will be amplified and exacerbated in the “poorer” solvent ethanol, resulting in a less homogeneous gel.

Conclusions We have demonstrated that increasing the extent of crosslinks in a solvent-swollen gel can result in a corresponding increase in the diffusion coefficient of a small solute dissolved in that gel. This phenomenon, which reflects a cross-link-dependent increase in the heterogeneity of the gel, appears to arise as a consequence of the solvent used to swell the polymer. Further experiments are being performed to elaborate upon and substantiate this model. These results are seen to be particularly pertinent in the study of intracellular events that occur upon photoinduced cell death. For example, upon the photosensitized production of intracellular singlet oxygen, one response of the cell is the collapse of one of the cytoskeletal proteins into more dense, inhomogeneously distributed domains.19,42 Thus, one disrupts a hydrogel that appears somewhat homogeneous (i.e., the viable cell), creating an intracellular medium through which solute diffusion can be substantially altered.

Experimental Section Sample Preparation. Glutaraldehyde-cross-linked PVA samples were prepared using an established technique.1 Briefly, 2.0 g of PVA (31 000-50 000 g/mol, 98-99% hydrolyzed, Aldrich) was added to 18 g of distilled water and maintained at 80 °C for 90 min with stirring. Thereafter, this PVA solution was cooled to room temperature before adding the glutaraldehyde-based cross-linking mixture. For a cross-linking degree of 0.01 [defined as (# of crosslinking molecules)/(# of polymer repeat units)], the latter consisted of 4 mL of water, 171 µL of glutaraldehyde solution (25% w/w in water, Aldrich), 80 µL of 98.5% H2SO4 as a cross-linking catalyst, 169 µL of 100% acetic acid as a buffer, and 1.2 mL of methanol as a cross-linking quencher. For different cross-linking degrees, the amount of added glutaraldehyde solution was changed. In these latter cases, although the amounts of the other additives remained the same, the amount of added water was adjusted such that the total reaction volume remained the same. Upon addition of the cross-linking mixture to PVA, the solution was stirred for 1 min and then sonicated for 30 s to remove air bubbles. Using pipets, a suitable amount of the mixture was then transferred to an 8 cm diameter glass Petri dish, covered, and allowed to stand for 48 h at room temperature. Over this period, the gel produced with the least amount of cross-linking mixture (i.e., crosslinking degree of 0.01) retained the 8 cm diameter of the Petri dish. (42) Can˜ete, M.; Lapen˜a, M.; Juarranz, A.; Vendrell, V.; Borrell, J. I.; Teixido´, J.; Nonell, S.; Villanueva, A. Anti-Cancer Drug Des. 1997, 12, 543–554.

Schack et al. Table 3. Extent to Which the Cross-Linked PVA Samples Shrunk When Water Was Replaced by Ethanol as the Swelling Solvent degree of cross-linkinga

% change in sample diameter

0.010 0.020 0.050 0.067 0.100

40 40 26 5 0

a

(# of cross-linking molecules)/(# of polymer repeat units).

For the remaining samples prepared with more cross-linking, the gel shrunk by an amount that increased with the amount of added crosslinker. This phenomenon has likewise been previously observed.1 Upon visual examination, samples with cross-linking degrees of 0.01 and 0.02 appeared clear. The remaining samples appeared slightly cloudy. The resultant gels were washed with water repeatedly over 48 h. The gels were then repeatedly exposed to 96% ethanol over a period of 48 h to ensure complete solvent exchange. The change in solvent from water to ethanol had a noticeable effect on the size of the least cross-linked gels; these four samples shrunk by an appreciable amount (Table 3). With this change in solvent, all samples now appeared slightly cloudy. Moreover, upon physically handling these materials, the relative elasticity of the gels decreased noticeably with an increase in the cross-linking degree. For the oxygen diffusion measurements, the edges of the pancakeshaped ethanol-swollen gels were removed to help ensure a sample of uniform thickness. Depending on the sample, the thickness ranged from 270 ( 10 to 1250 ( 10 µm, as measured using a digital thickness gauge (Mitotoyo Digimatic Indicator, model ID-C1012EB). Sample thickness was most efficiently measured by placing the gel between two glass plates, measuring the thickness of this sandwich, and then subtracting the thickness of the glass plates. Sensitizer Incorporation. For samples containing the singlet oxygen sensitizer TMPyP, the gel was simply exposed to an ethanol solution containing TMPyP for 48 h. Attempts were made to adjust the concentration of TMPyP to yield an absorbance of ∼0.5 at 421 nm (i.e., TMPyP Soret band maximum) over the path length of that given sample. However, since thickness varied from one sample to the next, a final absorbance of ∼0.5 was most easily achieved by tuning the excitation wavelength to an appropriate position on the Soret band, for example between ∼445 and 455 nm. TMPyP (Aldrich, product no. 323497) was used as received. Absorption spectra obtained indicate that TMPyP does not aggregate in our samples. Moreover, visual inspection of the TMPyPcontaining gels suggests that this dye is homogeneously distributed throughout the sample. Given the way our experiments are done, these observations, in themselves, are sufficient to validate our approach. Nevertheless, TMPyP may still be inhomogeneously distributed in our gel on a length scale that is coincident with the inhomogeneities identified in our SAXS experiments (i.e., length scale of ∼2-12 nm). In light of this, it is important to recognize that the molecule actually probed, singlet oxygen, is mobile and will diffuse over a given distance within its lifetime. If we assume an oxygen diffusion coefficient D of 1.3 × 10-6 cm2 s-1 and a singlet oxygen lifetime of 13 µs (i.e., that in ethanol), then the root-meansquare radial distance traveled, r, in a period of two lifetimes (i.e., t ) 26 µs) is 142 nm [i.e., r ) (6Dt)1/2]. Thus, this diffusion distance would clearly mask any effect of sensitizer inhomogeneity. Photosensitized Singlet Oxygen Experiments. The ethanolswollen gels were placed into the sample chamber shown in Figure 1. The system was designed such that the perimeter of the gel pushed up against a shelf in the chamber. This ensured that all of the sorbed oxygen entered only through one face of the gel. In a typical experiment, nitrogen gas, presaturated with ethanol, was bubbled into the chamber until the intensity of the 1270 nm singlet oxygen emission signal reached a minimum. At this point, the ethanol surrounding the gel was rapidly exchanged using a syringe coupled to the chamber with HPLC fittings. The injected ethanol had been

Oxygen Diffusion in Ethanol-Swollen PVA Gels saturated with a mixture of nitrogen and oxygen gas (1% oxygen). Upon such exposure of the gel to ambient oxygen, the 1270 nm emission signal increased in intensity, yielding a sorption curve such as that shown in Figure 2. Rheology Experiments. The viscoelastic properties of our gels were examined using a rotational rheometer (Haake MARS) with a parallel plate geometry (double serrated, PP35/s cell, 35 mm diameter). Experiments were performed at 25 °C, and the ethanolswollen samples were examined in an ambient ethanol environment. The gels were subjected to periodic oscillations over the frequency range 0.1-10 Hz, evaluating both the elastic response (i.e., storage modulus, G′) and the viscous response (i.e., loss modulus, G′′).33,34 Values of these respective moduli at 1 Hz were compared. An independent control experiment was first performed to ascertain what stress could be applied to the gels in the frequency scans. A stress scan was performed over the range 0.1-1000 Pa at a frequency of 1 Hz. Linear viscoelastic response was observed with an applied stress of up to 15 Pa, and, as such, the viscoelastic moduli were determined with an applied stress of 8 Pa. Small-Angle X-ray Scattering (SAXS) Experiments. Experiments were performed using instrumentation and an approach that have been previously described.43 The ethanol-swollen gels were cut and inserted into a quartz capillary tube, and ethanol was then (43) Pedersen, J. S. J. Appl. Crystallogr. 2004, 37, 369–380.

Langmuir, Vol. 25, No. 2, 2009 1153 added to the capillary tube to ensure swelling to equilibrium. The SAXS signal obtained from ethanol was used as a background and subtracted from the signal obtained from the gel. The scattering intensity is obtained as a function of the scattering vector modulus q ) (4π/λ)sin θ, where λ is the X-ray wavelength and 2θ is the scattering angle. For a given sample, we do not know the exact concentration of polymer in the X-ray beam. However, at high scattering vectors, q, the intensity originates from the polymer strands. Therefore, at high q, one has a signal that scales with the polymer concentration. As such, all data were normalized so that they agree at high q, allowing us to ascribe meaning to relative changes in the scaling factors seen in eq 2.

Acknowledgment. This work was supported by the Danish National Research Foundation through a block grant for the Center for Oxygen Microscopy and Imaging. N.B.S. thanks the Danish Graduate School of Polymer Science for partial support of his Ph.D. study. Supporting Information Available: Additional comments on SAXS interpretation. This material is available free of charge via the Internet at http://pubs.acs.org. LA803024H