Langmuir 2006, 22, 2241-2248
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Gels of Hydrophobically Modified Hydroxyethyl Cellulose Cross-Linked by Amylose. Competition with Cyclodextrin Maria Karlberg, Lennart Piculell,* and Sylvaine Ragout Physical Chemistry 1, Center for Chemistry and Chemical Engineering, Lund UniVersity, P.O. Box 124, SE-221 00 Lund, Sweden ReceiVed August 10, 2005. In Final Form: December 20, 2005 Previous work has shown that amylose (AM) can cross-link hydrophobically modified polymers by inclusion complexation, whereby thermoreversible cold-setting gels are formed. In this work, the competition between AM and cyclodextrin (CD) for the formation of inclusion complexes with hydrophobically modified hydroxyethyl cellulose (HMHEC) is investigated. A detailed study of viscosity, NMR self-diffusion, and chemical shifts of the two-component mixture, CD and HMHEC, was performed. The results imply that 2:1 (CD:polymer hydrophobe) complexes may be formed. The three-component mixtures, HMHEC/AM/CD, were investigated by rheology, NMR self-diffusion, and intensities of the NMR resonance peaks. The CD molecules competed efficiently with the AM molecules, as seen by a decreased storage modulus, an increased self-diffusion of AM and HMHEC, and increased NMR intensities of the HMHEC hydrophobes, as the concentration of CD increased in the solution. A high concentration of CD is needed in the mixtures to inhibit all interactions between HMHEC and AM, and it was shown that there still is an effect of AM at excess CD concentration in the mixtures.
Introduction A hydrophobically modified polymer (HMP) consists of a hydrophilic backbone to which a small amount of hydrophobes, typically short hydrocarbon chains, has been grafted. The polymers act as amphiphiles in water solution since they contain both hydrophobic and hydrophilic groups. In semidilute solutions, the hydrophobic groups aggregate intermolecularly to minimize their contact with water, and a three-dimensional network is thus formed. Amylose (AM) is the essentially unbranched fraction of starch, consisting of R-D-(1-4) glycosidic bonds. It is well-known that the AM can form inclusion complexes with amphiphilic compounds such as iodine1-4 and surfactants.5-14 In a complex, the hydrophobic part of the guest molecule is inserted into the central, hydrophobic cavity of the AM helices. Recent investigations have shown that amylose also can form inclusion complexes with polymer hydrophobes, whereby mixed gels are formed.15-19 The gelation has been attributed to the formation of long-lived cross-links where one AM molecule * Author to whom correspondence should be addressed. E-mail:
[email protected]. (1) Calabrese, V. T.; Khan, A. J. Polym. Sci., Part A: Polym. Chem. 1999, 37, 2711. (2) Knutson, C. A. Carbohydr. Polym. 1999, 42, 65. (3) Barrett, A. J.; Barrett, K. L.; Khan, A. J. Macromol. Sci., Pure Appl. Chem. 1998, 35, 1603. (4) Yamamoto, M.; Takayuki, S.; Yasunga, T. Bull. Chem. Soc. Jpn. 1982, 55, 1886. (5) Egermayer, M.; Piculell, L. J. Phys. Chem. B 2003, 107, 14147. (6) Gunning, P. A.; Giardina, T. P.; Faulds, C. B.; Juge, N.; Ring, S. G.; Williamson, G.; Morris, V. J. Carbohydr. Polym. 2003, 51, 177. (7) Lundqvist, H.; Eliasson, A.-C.; Olofsson, G. Carbohydr. Polym. 2002, 49, 43. (8) Lundqvist, H.; Eliasson, A.-C.; Olofsson, G. Carbohydr. Polym. 2002, 49, 109. (9) Svensson, E.; Gudmundsson, M.; Eliasson, A.-C. Colloids Surf., B 1996, 6, 227. (10) Eliasson, A.-C. Thermochim. Acta 1994, 246, 343. (11) Yamamoto, M.; Harada, S.; Nakatsuka, T.; Sano, T. Bull. Chem. Soc. Jpn. 1988, 61, 1471. (12) Kowblansky, M. Macromolecules 1985, 18, 1776. (13) Yamamoto, M.; Sano, T.; Harada, S.; Yasunaga, T. Bull. Chem. Soc. Jpn. 1983, 56, 2643. (14) Takagi, T.; Isemura, T. Bull. Chem. Soc. Jpn. 1960, 33, 437.
participates in inclusion complexes with several polymer hydrophobes. Recently, effects of added surfactants on mixed gels of AM and hydrophobically modified hydroxyethyl cellulose (HMHEC) have been studied.16 Surfactants added to the gel could destroy the AM/HMHEC complexation. The surfactants can compete with the AM/HMHEC complex in two ways. They can form inclusion complexes with AM and they can form mixed hydrophobic aggregates with the polymer hydrophobes. In the present investigation, we use competition experiments with added cyclodextrins (CD) as a means to obtain indirect information about the AM/HMHEC complexation. CD:s are cyclic oligosaccharides of R-D-glucose linked by R-(1-4) glucosidic bonds. A CD molecule corresponds closely to one turn of the AM single helix20 and, like AM, CD molecules can form inclusion complexes with various hydrophobic substances, such as HMP:s, surfactants, lipids, and alcohols.21-23 The simplest inclusion complex consists of one cyclodextrin and one guest molecule. Depending on the length of the hydrophobic chain, complexes with two CD molecules may form. Adding CD to an HMP solution results in reduced viscosity since the CD molecules form inclusion complexes with the polymer hydrophobes, which thereby become deactivated and cannot contribute to the threedimensional polymer network.24-29 (15) Egermayer, M.; Karlberg, M.; Piculell, L. Langmuir 2004, 20, 2208. (16) Egermayer, M.; Norrman, J.; Piculell, L. Langmuir 2003, 19, 10036. (17) Chronakis, I. S.; Egermayer, M.; Piculell, L. Macromolecules 2002, 35, 4113. (18) Gruber, J. V.; Konish, P. N. Macromolecules 1997, 30, 5361. (19) Okaya, T.; Kohno, H.; Terada, K.; Sato, T.; Maruyama, H.; Yamauchi, J. J. Appl. Polym. Sci. 1992, 45, 1127. (20) Immel, S.; Lichtenthaler, F. W. Sta¨rke 2000, 52, 1. (21) Del Valle, E. M. M. Process Biochem. 2004, 39, 1033. (22) Connors, K. A. Chem. ReV. 1997, 97, 1325. (23) Loftsson, T.; Brewster, M. E. J. Pharm. Sci. 1996, 85, 1017. (24) Horsky, J.; Mikesova, J.; Quadrat, O. J. Rheol. 2004, 48, 23. (25) Liao, D.; Dai, S.; Tam, K. C. Polymer 2004, 45, 8339. (26) Abdala, A. A.; Tonelli, A. E.; Khan, S. A. Macromolecules 2003, 36, 7833. (27) Karlson, L.; Thuresson, K.; Lindman, B. Langmuir 2002, 18, 9028. (28) Karlson, L.; Thuresson, K.; Lindman, B. Carbohydr. Polym. 2002, 50, 219. (29) Zhang, H.; Hogen-Esch, T. E. Langmuir 1998, 14, 4972.
10.1021/la052177i CCC: $33.50 © 2006 American Chemical Society Published on Web 01/28/2006
2242 Langmuir, Vol. 22, No. 5, 2006
In this investigation, we mostly use methylated R-CD (six glucose units), M-R-CD, and, in addition, β-CD (seven glucose units) for comparison in selected experiments. We start by investigating the viscosity, the chemical shift, and the selfdiffusion of the two-component system, HMHEC and M-R-CD. The paper then continues with the more complex system where AM is added as a third component and the competition between CD and AM is studied by rheology, intensity of the NMR resonance peaks, and diffusion. To enable direct comparisons with previous studies from our laboratory, we have used the same type of HMHEC/AM mixtures as in refs 16 and 17 and the same CD samples as in ref 28. Experimental Section Materials. Potato amylose (AM) with a molecular weight of about 800 000 g/mol was obtained from Sigma Chemical Co. Prior to use, the AM was placed in an oven at 80 °C for 1 h to minimize solvent impurities such as butanol. (Butanol is used as a precipitant in the extraction of AM from starch.) Hydrophobically modified hydroxyethyl cellulose, HMHEC, with the commercial name Natrosol Plus grade 331 was obtained from Aqualon. According to the manufacturer, the HMHEC has an average molecular weight of 250 000 g/mol and the degree of substitution of hydroxyethyl groups per repeating anhydroglucose unit equals 3.3. The polymer material was analyzed according to the method described by Landoll30 and was found to contain 0.765 wt % C16 alkyl chains of the dry sample weight. This corresponds to 0.34 mM alkyl chains in a 1 wt % aqueous polymer solution. The HMHEC is the same as has previously been used in complexation investigations with amylose.16,17 Prior to use, HMHEC was dissolved in water to a concentration of 1 wt %. Low molecular impurities, such as salt, were removed by dialysis against Millipore water in a Filtron Ultrasette device. The dialysis was performed until the expelled water showed a conductivity of less than 2 µS/cm. The polymer was then freeze-dried. In the present investigation, the HMHEC concentration was 1 wt %, which is well above the critical overlap concentration (ca. 0.2 wt % for unmodified HEC). Methylated R-cyclodextrin (M-R-CD), consisting of six glucose units, was supplied by Wacker-Chemie under the trade name Cyclodextrin Alpha W6 M1.8. The degree of methylation per glucose unit, as given by the supplier, was 1.6-1.9. The M-R-CD sample was the same as that used by Karlson et al.27,28,31 in their investigations. The compound was of pharmaceutical quality and was used without further purification. β-Cyclodextrin (β-CD), consisting of seven glucose units, was supplied by Wacker-Chemie under the trade name CavamaxW7 Pharma. Water of Millipore quality (resistivity ∼ 18 MΩ cm-1) was used for the rheology samples, and deuterium oxide (99.8 atom-% D) obtained from Dr. Glaser AG (Basel, Switzerland) was used for the NMR samples. Sample Preparation. Stock solutions of HMHEC were prepared at room temperature by mixing and stirring with a magnetic stirrer. AM samples were prepared at appropriate concentrations, were heated to 150 °C for approximately 20 min in a Pierce Reacti-Therm heating/ stirring module, were cooled to 90 °C, and finally were added to the prepared room-temperature HMHEC solutions. The mixed gels were cooled to 25 °C before addition of CD. Appropriate amounts of CD stock solution at room temperature (or water in the samples without CD) were added to the gels or to the AM-free HMHEC solution, as appropriate. Rheology and Viscometry. Oscillatory measurements were performed on mixtures with AM on a controlled stress Physica UDS 200 rheometer using a cone-and-plate geometry (50 or 70 mm radius, 1°). All measurements were performed within the linear viscoelastic region, which was checked for each sample. The storage modulus (G′) and the loss modulus (G′′) were measured as functions of the (30) Landoll, L. M. J. Polym. Sci., Part A: Polym. Chem. 1982, 20, 443. (31) Karlson, L.; Malmborg, C.; Thuresson, K.; So¨derman, O. Colloids Surf., A 2003, 228, 171.
Karlberg et al. angular frequency of oscillation, ω, in the range of 0.0628-50.27 rad/s (corresponding to 0.01-8 Hz). Flow experiments were performed on samples with low viscosity. The experiments were performed on a controlled stress CarriMed CSL 100 rheometer using a cone-and-plate geometry (60 mm radius, 1°) in the range 0.61000 s-1. Each sample was placed on the plate held at 25 °C and was allowed to equilibrate for at least 30 min prior to the measurements. NMR Spectroscopy. Proton NMR measurements were performed at 27 °C on a Bruker Fallanden (Switzerland) model ARX500 spectrometer, operating at 500.25 MHz. Pulsed field gradient (PFG) 1H NMR experiments were performed at 25 °C on a 200 MHz Bruker DMX spectrometer equipped with a Bruker DIFF-25 gradient probe driven by a Bruker BAFPA-40 unit. The chemical shifts are given relative to tetramethylsilane (TMS), and the resonance peak of the residual protons of the solvent is then at 4.7 ppm. The self-diffusion coefficients of M-R-CD, HMHEC, and AM were obtained by using pulse-gradient spin-echo proton NMR (1HPGSE). The gradient strength, G, was varied in the range 0.5-9 T/m. The diffusion time, ∆, was in the range 25-500 ms and the duration of the gradient pulses, δ, was in the range 0.5-2 ms. The HMHEC and the AM resonance peaks were overlapped with resonance peaks from M-R-CD. The diffusion coefficients of HMHEC and AM were therefore evaluated using the CORE software.32 Owing to its inherent polydispersity, a polymer sample generally contains a distribution of molecular species with different diffusion coefficients. However, the overlapping resonance peaks in the present case do not allow a full characterization of these distributions. We therefore chose to describe the diffusion of HMHEC in terms of two components (“slow” and “fast”) in our analysis, and we report both of the corresponding diffusion coefficients below. For AM, we only found it meaningful to extract one effective diffusion coefficient, owing to the much weaker signal. One of the CD resonance peaks (∼5.13 ppm) was well separated from the peaks of the HMHEC spectrum, and the decay of this peak was possible to follow when the M-R-CD concentration was higher than 1 mM. The resolution of the diffusion measurements at lower M-R-CD concentrations was poor and no reliable diffusion coefficients could be obtained for M-R-CD in these mixtures. In mixtures with AM, the M-R-CD resonance peaks were unfortunately disturbed by the presence of AM and no accurate diffusion coefficients for M-R-CD could be obtained. The diffusion coefficient for M-R-CD was evaluated by assuming one-component diffusion (rapid exchange between all environments experienced by the molecules) and monodisperse species. The attenuation of the signal intensities E(k) in the NMR diffusion experiment is then given by33 E(k) ) exp(-kD)
(1)
where D is the self-diffusion coefficient and k ) (γGδ)2(∆ - δ/3)
(2)
where γ is the gyromagnetic ratio of the spin-bearing nucleus (γ ) 2.6752 × 108 rad T-1s-1), and the pulse sequence parameters are defined above. In all cases, the experimentally obtained signal attenuations for M-R-CD were found to be well described by a single-exponential decay according to eq 1.
Results and Discussion Addition of CD to HMHEC Alone. Before studying the competition of CD with the AM-HMHEC complex, we made a detailed characterization of the interaction between CD and HMHEC using viscometry and NMR spectroscopy. Viscosity. The viscosity of an aqueous solution of HMHEC decreases as CD is added to the solution. The samples were shear-thinning, and this behavior was most pronounced for the (32) Stilbs, P.; Paulsen, K.; Griffiths, P. C. J. Phys. Chem. 1996, 100, 8180. (33) Stejskal, E. O.; Tanner, J. E. J. Chem. Phys. 1965, 42, 288.
Gels of Modified Cellulose Cross-Linked by Amylose
Figure 1. Relative viscosity of 1 wt % HMHEC as a function of M-R-CD concentration. The full line represents the fit of the data to eq 3 when the number of binding sites is 0.35 mM and the complexation constant is 7.5 mM-1.
samples with an M-R-CD concentration below 1 mM M-R-CD. For these samples, the viscosities did not reach a true Newtonian plateau, on a log-log plot, in the accessible frequency range, but they nevertheless clearly leveled off at low shear rates. The viscosity values shown in Figure 1 are therefore the apparent viscosities at 10 s-1, but the same trend of decreasing viscosity with increasing M-R-CD concentration was seen independent of the shear rate. The viscosity in Figure 1 levels off above ca. 3 mM M-R-CD. It is of interest to compare the results in Figure 1 with the viscosity data obtained by Karlson et al. for the addition of the same M-R-CD to solutions of hydrophobically modified ethyl(hydroxy ethyl) cellulose (HMEHEC).28 We have therefore analyzed our data using the simple quantitative model proposed by the previous authors. In this model, it is assumed that the zero-shear viscosity can be expressed as the product of the limiting storage modulus at high frequencies, G∞, times the characteristic time of the relaxation process. G∞ is proportional to the crosslinking density, and the characteristic relaxation time is assumed to be the (constant) lifetime of a hydrophobe in an aggregate. The viscosity then becomes proportional to the cross-linking density. Effects due to entanglements of the polymer backbone are neglected. It is also assumed that 1:1 complexes between CD and the polymer hydrophobes are formed and that the binding sites are independent. The concentration of binding sites is expected to be the same as the number of polymer hydrophobes, and a hydrophobe complexed with a CD molecule is considered as inactive. From these considerations, it follows that
η - η∞ )1η0 - η ∞ B + ctot,CD + 1/K 2
x
(B + ctot,CD + 1/K)2 - BcCD 4 (3) B
where ctot,CD is the total CD concentration in the sample, η is the viscosity of the mixture, η0 and η∞ are the viscosities without and at excess CD, respectively, B is the number of binding sites, and K is the complexation constant. The detailed derivation of eq 3 is given in ref 28. The complexation constant and the number of binding sites have been varied independently to obtain the best fit to the experimental data. The best fits (see Figure 1) were obtained when the number of binding sites was less than 0.5 mM and the complexation constant was in the range 5.0-10.0 mM-1. The number of binding sites obtained from the fit (see Figure 1) is comparable to the number of hydrophobes, 0.34 mM, obtained by chemical analysis (see the Experimental Section).
Langmuir, Vol. 22, No. 5, 2006 2243
Figure 2. Self-diffusion coefficients of the slow (squares) and the fast (triangles) component of HMHEC and M-R-CD (circles) at increasing M-R-CD concentration in 1 wt % solutions of HMHEC. The horizontal line corresponds to the self-diffusion of M-R-CD in 1 wt % unmodified HEC.
In their investigation of the binding of the same M-R-CD to HMEHEC, Karlson et al.28 obtained a similarly good agreement between the concentration of hydrophobes and the concentration of binding sites obtained from viscosity using eq 3. However, they obtained a significantly higher complexation constant of 44.0 mM-1, despite the fact that their EHEC was modified with shorter (C14) alkyl chains than the C16 chains of our HMHEC. Previous studies on simpler surfactant systems show that the complexation constant for a cyclodextrin increases with increasing alkyl chain length, as expected.34-36 We speculate that the low apparent binding constant obtained in our analysis is because the 1:1 complexes with C16 chains are not rheologically inactive, as assumed in the simple model. One cyclodextrin molecule cannot contain the entire polymer hydrophobe,37 and it is possible that the part that is not covered by cyclodextrin may still provide hydrophobic cross-linking. This means that 2:1 complexes between M-R-CD and the polymer hydrophobes would be required to yield completely rheologically inactive hydrophobes. The lower complexation constant obtained in our analysis would then correspond to an effective constant influenced by both the first and the second complexation steps of the M-R-CD molecules. In this context, the small deviations between the experimental data and the simple model (assuming only 1:1 complexation) seem to be systematic; see Figure 1. An inclusion of rheologically active 1:1 complexes in the viscosity model was not attempted, however, since this would make the model excessively complicated. In particular, one cannot assume that the lifetime in an aggregate is the same for a 1:1 complex as for an uncomplexed hydrophobe. Diffusion. The self-diffusion of M-R-CD and HMHEC, respectively, is shown in Figure 2. The self-diffusion of M-RCD and the fast and the slow components of HMHEC increase as the connectivity of the network decreases with increasing M-R-CD concentration. The self-diffusion coefficients level off above ca. 3 mM M-R-CD, which agrees well with the leveling off of the viscosity in Figure 1. When leveling off, the selfdiffusion coefficient of M-R-CD reaches the self-diffusion of M-R-CD in a solution of unmodified HEC. By using the self-diffusion coefficient of M-R-CD in unmodified HEC together with the diffusion data in Figure 2, it is possible to extract the concentration of bound M-R-CD molecules using the well-known relation valid for a rapid exchange (on the time scale of the diffusion time ∆) between bound and free cyclodextrin:38 (34) Mwakibete, H.; Bloor, D. M.; Wyn-Jones, E. Langmuir 1994, 10, 3328. (35) Park, J. W.; Song, H. J. J. Phys. Chem. 1989, 93, 6454. (36) Wilson, L. D.; Verall, R. E. Can. J. Chem. 1998, 76, 25. (37) Funasaki, N.; Ishikawa, S.; Neya, S. J. Phys. Chem. B 2003, 107, 10094.
2244 Langmuir, Vol. 22, No. 5, 2006
DCD,obs ) PCD × DHMHEC + (1 - PCD) × DCD,f
Karlberg et al.
(4)
where DCD,obs is the observed M-R-CD diffusion, PCD is the fraction of M-R-CD bound to HMHEC, DHMHEC is the HMHEC diffusion, and DCD,f is the diffusion of free M-R-CD. The concentration of bound M-R-CD molecules, cb,CD, can then be calculated as
cb,CD ) PCD × ctot,CD
(5)
where ctot,CD is the total CD concentration in the sample. The diffusion of free M-R-CD can be assumed to be in the interval between the diffusion of M-R-CD in water (2.5‚10-10 m2 s-1) and the diffusion of M-R-CD in 1 wt % unmodified HEC (2.0‚10-10 m2 s-1). The diffusion of bound M-R-CD can be considered to be the same as the diffusion of HMHEC. In the calculations, the self-diffusion coefficient of HMHEC was put equal to zero without loss of accuracy, since the first term in eq 4 was completely negligible also when the self-diffusion coefficient of the fast component of HMHEC was used. These assumptions gave the limits 0.45-0.76 mM bound M-R-CD molecules when the total M-R-CD concentration was 2.2 mM. This concentration of bound cyclodextrin is significantly higher than the concentration of polymer hydrophobes (0.34 mM) and thus supports the notion that 2:1 complexes are formed. It was found impossible to deduce whether the concentration of bound M-R-CD leveled off at higher M-R-CD concentrations since the uncertainty in the difference between DCD,obs and DCD,f then became too large. NMR Shift Data. Information complementary to the diffusion and the viscosity measurements can be obtained from the HMHEC hydrophobe resonance peaks in the NMR spectra. These spectra are sensitive to local properties of the molecules and, thus, provide another tool to see if 2:1 complexes are present at high M-R-CD concentrations. Figure 3 shows the spectra of 1 wt % HMHEC alone and in mixture with 0.11, 1.1, and 9.9 mM M-R-CD. The polymer hydrophobe peaks are shifted downfield as the M-RCD concentration increases in the solution. The resonance peaks from M-R-CD (not shown) are shifted upfield with increasing concentration, but the peaks from the polymer hydrophobes are shifted the most. The chemical shift changes when mixing surfactants and CD have been investigated before.37,39-42 The surfactant chemical shift has been found to level off in some cases36 and to go through a maximum36,39 as 2:1 complexes are formed. Starting at 0.5 mM M-R-CD, the originally overlapping peaks from the intermediate methylene groups in the polymer hydrophobe split up into two peaks, which are shifted differently with increasing M-R-CD concentration. Both peaks are broadened and shifted as the M-R-CD concentration increases. A splitting of peaks in mixtures of CD and surfactants has been observed before and the literature contains different explanations. It has been suggested that the splitting may be due to slow exchange of the CD molecules between the complexed state and free CD molecules.42 It has also been proposed that the splitting appears because of formation of 1:1 and 2:1 complexes, giving rise to different chemical shifts.36 (38) Evans, D. F.; Wennerstro¨m, H. The Colloidal Domain where Physics, Chemistry, Biology and Technology Meet, 2nd ed.; Wiley-VHC: New York, 1999; Chapter 4. (39) Funasaki, N.; Ishikawa, S.; Neya, S. J. Phys. Chem. B 2004, 108, 9593. (40) Lu, R.; Hao, J.; Wang, H.; Tong, L. J. Inclusion Phenom. Macrocyclic Chem. 1997, 28, 213. (41) Fielding, L. Tetrahedron 2000, 56, 6151. (42) Abrahmsen-Alami, S.; Alami, E.; Eastoe, J.; Cosgrove, T. J. Colloid Interface Sci. 2002, 246, 191.
Figure 3. Partial 1H NMR spectra of HMHEC with added M-R-CD from bottom to top: 1 wt % HMHEC alone, with 0.11, 1.1, and 9.9 mM M-R-CD. Well-resolved peaks appear from the polymer hydrophobes. Without added M-R-CD, the shifts are methyl at 0.80 ppm, intermediate methylene groups at 1.21 ppm, and β-methylene protons at 1.50 ppm. The origin of the resonance peaks at 1.07 and 1.15, which remain unaffected by added cyclodextrin, is unknown.
In our mixtures, none of the two methylene peaks have the same chemical shift as the hydrophobes alone and both shifts increase continuously with increasing concentration of M-RCD. This indicates that fast exchange occurs and that the two peaks cannot be due to one population of free polymer hydrophobes and one population of complexed hydrophobes or one with 1:1 complexes and one with 2:1 complexes. These situations would, contrary to our results, have resulted in peaks at constant shifts, but with changing intensities, at increasing cyclodextrin concentration. One possible interpretation of the splitting would be that the HMHEC sample contains two distinct groups of hydrophobes which differ in their ability to form complexes with M-R-CD. We see no reason to invoke such a bimodal distribution, however. Moreover, if two groups of polymer hydrophobes existed, all hydrophobe resonance peaks, including the methyl peak at 0.8 ppm, should be split, but here only the methylene peaks are affected. A more probable explanation is that there is only one class of hydrophobes but that there are two categories of methylene groups on a complexed hydrophobe. One M-R-CD molecule only covers about half the methylene groups on the hydrophobes, and two distinct peaks, as opposed to a smooth distribution, imply, in line with the suggestions of Abdala et al.,26 that the M-R-CD molecule is situated more often at a certain part of the polymer hydrophobe because of short-range interactions. These methylene groups give rise to one part of the peak. The bound M-R-CD molecule then occasionally diffuses along the polymer hydrophobe, giving rise to the observed shift of the second part of the peak. The changes in chemical shift with added M-R-CD are shown in Figure 4. The chemical shift data can be used to obtain the complexation constant between the CD and the polymer hydrophobes. We will attempt an analysis using the simplest possible model, in the same spirit as in the analysis of the viscosity data above. The model used here has previously been used to investigate the complexation between surfactants and CD43,44 (43) Cabaleiro-Lago, C.; Nilsson, M.; So¨derman, O. Lamgmuir 2005, 21, 11637. (44) Valente, A. J. M.; Nilsson, M.; So¨derman, O. J. Colloid Interface Sci. 2005, 281, 218.
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Langmuir, Vol. 22, No. 5, 2006 2245
Figure 4. Variations in the chemical shifts as a function of the M-R-CD concentration. The full lines are the fits obtained with the model described in the text. (a) The resonance peak initially at 0.80 ppm, giving the complexation constant 3.1 ( 0.17 mM-1, (b) the resonance peak initially at 1.50 ppm, giving the complexation constant 2.0 ( 0.59 mM-1, (c) one part of the peaks initially at 1.21 ppm, giving the complexation constant 8.3 ( 5.4 mM-1, and (d) the other part of the peak initially at 1.21 ppm, giving the complexation constant 8.3 ( 4.0 mM-1.
and assumes that a 1:1 complex is formed between a CD and a polymer hydrophobe (HP).
CD + HP T CD - HP
(6)
The stability of the complex can be described in terms of a complexation constant, defined as
K ) [CD - HP]/([CD][HP])
(7)
The chemical shift of the polymer hydrophobes can be expressed as
∂HP,obs ) ∂CD-HP(1 - PHP,f) + ∂HP,f × PHP,f
(8)
where ∂obs is the observed chemical shift of the polymer hydrophobes, ∂CD-HP is the chemical shift of hydrophobes which are in complex with CD, ∂HP,f is the shift of hydrophobes which are not in complex, and PHP,f is the fraction of hydrophobes which are not in complex. It is assumed that fast exchange occurs. One fit for each set of shift data is then performed in which the best values of ∂HP,f, ∂CD-HP, and K are determined. The results are given in Figure 4. The increase in chemical shift is most pronounced at low M-R-CD concentrations and continues to increase slowly in the whole investigated M-R-CD concentration range. The chemical shift data do not reach a plateau, which indicates that additional complexes of polymer hydrophobes and M-R-CD are formed even after the viscosity and the self-diffusion coefficients have leveled off. The method applied does not provide a perfect fit of the shift data and systematic deviations are apparent, especially for the methylene peaks. The (apparent) 1:1 complexation constants obtained from the various fits are about 1-10 mM-1 (see Figure 4), which may be compared with the values reported in the literature for CD and surfactants with the same hydrophobic
chain length as HMHEC; these were 110 mM-1 for the first CD and 1.6 mM-1 for the second CD when R-CD34 was used and were in the ranges 56-90 mM-1 and 0.002-1.5 mM-1, respectively, for the first and the second CD when β-CD34,35 was used. We propose that the low apparent complexation constants obtained in our analysis correspond to an effective constant influenced by both the first and the second complexation steps of the M-R-CD molecules, in line with the complexation constant obtained in the viscosity investigation. The lower complexation constant in comparison with surfactants could possibly also be due to difficulties of complex formation because of the stiffness and the bulkiness of the polymer backbone. This is a less likely explanation, however, since a complexation constant of 44.0 mM-1 has been obtained with a closely similar polymer and the same M-R-CD.28 Again, we do not find it meaningful to include 2:1 complexes explicitly in the shift model, since this would introduce two more parameters (one complexation constant and one shift value for 2:1 complexes). The above evidence for 2:1 complexes is admittedly indirect, but the spectra in Figure 3 contain one other feature that provides more direct evidence. The line shape of the methyl group varies in a nonmonotonic fashion with added cyclodextrin: Initially (with no cyclodextrin) it is broad, then it narrows sufficiently to reveal a resolved triplet, and then it broadens again. This sequence, indicating a nonmonotonic variation in the mobility of the methyl group, is the expected result of strongly aggregated hydrophobes in the absence of CD, less aggregated hydrophobes with protruding chain ends for 1:1 complexes, and finally, less mobile methyl groups again for hydrophobes containing two bound CD molecules per alkyl chain. Competition of CD with Amylose. The competition study was performed by first mixing the HMHEC and the AM by the procedure described in the Experimental Section. CD stock solution was then added when the HMHEC-AM gel had equilibrated at room temperature. Previous studies have shown a maximum in gel strength (without CD) for 1 wt % HMHEC with 0.25 wt % AM. In this study, we have therefore looked at the competition with CD at 0.25 wt % AM and have compared the result with gels with less (0.1 wt %) AM. Rheology. Mechanical spectra were obtained with increasing M-R-CD concentration for the various AM concentrations. Figure 5 shows the changes in the storage, and the loss moduli with 0.25 wt % AM. Without added M-R-CD, the mechanical spectrum shows the characteristic feature for a gel, that is, G′ is independent of frequency and is much higher than G′′. This result is consistent with previous findings on the same system in our laboratory.15-17 With increasing M-R-CD concentration, both G′ and G′′ decrease continuously and the features of the mechanical spectrum become less gellike. This must be due to complexation between the M-RCD molecules and HMHEC hydrophobes, thereby replacing the AM/HMHEC complexes. Figure 5 shows that G′ and G′′ cross when the M-R-CD concentration is 2.2 and 3.3 mM and that G′ dominates at low frequencies. This relation between G′ and G′′ is found in systems that display (at least) two well separated relaxation times, such as polymer melts.45 To illustrate this feature, we can compare our experimental result with the simple case of a double Maxwell model, where the amplitude of one of the Maxwell elements is (45) Mark, J. E.; Eisenberg, A.; Graessley, W. W.; Mandelkern, L.; Samulski, E. T.; Koenig, J. L.; Wignall, G. D. Physical Properties of Polymers, 2nd ed.; American Chemical Society: Washington, DC, 1993; Chapter 3.
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Figure 5. G′ (filled symbols) and G′′ (empty symbols) at 1 rads-1 of mixtures with 1 wt % HMHEC and 0.25 wt % AM without CD (diamonds) and with 0.11 (squares), 0.55 (triangles pointing up), 1.1 (triangles pointing down), 2.2 (circles), and 3.3 (right-angled triangles) mM M-R-CD.
varying. In a single Maxwell model,46 G′ and G′′ can be written as
τ2ω2 G′(ω) ) G∞ 1 + τ2ω2
(9)
τω G′′(ω) ) G∞ 1 + τ2ω2
(10)
where G∞ represents the plateau value of G′ at high frequencies, τ is the relaxation time, and ω is the angular frequency. The double Maxwell model consists of a sum of two elements, one with a long relaxation time (indicated by “s” for slow) and one with a short relaxation time (indicated by “f” for fast). A theoretical sequence for the double Maxwell model is shown in Figure 6, where G∞,s for the slowly relaxing element is decreased continuously. The angular frequency interval corresponding to our experimental values is indicated by the vertical lines in the figure. This model captures the essential features of the experimental rheological data. When G∞,s is high (Figure 6a) G′ is almost independent of frequency while G′′ passes through a minimum. As G∞,s decreases, the minimum in G′′ moves toward lower frequencies. Figure 6d shows a situation where the number of active chains have decreased so much that G′ and G′′ cross over in the experimental frequency range and G′ dominates at low frequencies. The existence of two relaxation processes thus seems clear from our experiments, and we should try to provide a physical interpretation of their nature. One simple possibility is that G∞,s for the slow relaxation corresponds to the number of active chains mainly created by AM cross-links; as the M-R-CD concentration increases, the number of active chains decreases and G∞,s decreases accordingly. The fast relaxation process would then presumably correspond to entanglements in the (un-cross-linked) semidilute polymer solution. Taking this analysis one step further, we may (46) Barnes, H. A.; Hutton, J. F.; Walters, K. An Introduction to Rheology; Elsevier: Amsterdam, 1989; Chapter 3.
Figure 6. G′ and G′′ as functions of the angular frequency for a double Maxwell model where the number of active chains, and thereby G∞ for one of the Maxwell elements, decreases when going from figure a to d. The relaxation times for the fast and slow processes are 0.03 and 30 s, respectively. G∞,f is kept fixed at 40 Pa, while the value of G∞,s varies in the order 30, 10, 5, and 1 when going from Figure a to d. The full line represents G′ and the broken line represents G′′. The vertical lines indicate the experimental angular frequency range in this work.
compare the number of active chains in the HMHEC/AM network with estimates from a simple model. The number of active chains can be calculated from the theory of rubber elasticity (assuming that the free-energy change of chain deformation is purely entropic) through the relation
G∞ ≈ RTc
(11)
where R is the gas constant, T is the temperature, and c is the concentration of active chains. Equation 11 also contains a numerical prefactor of order unity, the exact value of which depends on the details of the network model. Using G∞,s ) 36 Pa, corresponding to the constant experimental G′ value for the mixture of HMHEC and AM without CD, eq 11 yields c ) 0.015 mM. This value is approximately 20 times lower than the concentration of HMHEC hydrophobes obtained by chemical analysis (see the Experimental Section), which represents an upper limit to the concentration of active chains. The low number of active chains obtained in the analysis may indicate either that some hydrophobes are sterically hindered to form inclusion complexes with AM or that a large number of elastically inactive loops are formed by the incorporation of many hydrophobes from the same HMHEC chain in a given AM/HMHEC cluster. A more complex model that should be considered is the “sticky reptation” model for concentrated networks of polymers with reversible cross-links developed by Leibler et al.47 This model also predicts two relaxation processes, where one corresponds to the lifetime of a cross-link (a “sticker”) and the other is the sticky reptation time of a polymer chain in a tube of surrounding chains, with which the polymer forms reversible cross-links. It is doubtful that this model is directly applicable to the complex HMHEC/AM gel, where the structure is much more complicated owing to the multifunctional cross-links presumably made up by (47) Leibler, L.; Rubinstein, M.; Colby, R. H. Macromolecules 1991, 24, 4701.
Gels of Modified Cellulose Cross-Linked by Amylose
Figure 7. The storage modulus at 1 rads-1 with increasing CD concentration in mixtures of 0.1 wt % AM with M-R-CD (circles), 0.25 wt % AM with M-R-CD (squares), and 0.25 wt % AM with β-CD (triangles).
Langmuir, Vol. 22, No. 5, 2006 2247
Figure 9. Self-diffusion coefficients of the fast component of HMHEC (circles), the slow component of HMHEC (triangles), and AM (crosses) with increasing M-R-CD concentration in mixtures with 1.0 wt % HMHEC without AM (filled symbols) and with 0.25 wt % AM (empty symbols).
Figure 8. The apparent viscosity at 10 s-1 at high M-R-CD concentrations of mixtures without (circles), with 0.1 wt % AM (squares), and with 0.25 wt % AM (triangles) in mixtures with 1.0 wt % HMHEC.
AM molecules with several hydrophobe inclusion complexes. In particular, the notion of an average tube seems doubtful. However, one insight that should carry over from the sticky reptation model is that one should be cautious to consider the slow relaxation time in the HMHEC/AM gel to be a direct measure of the lifetime of an AM-hydrophobe inclusion complex. The terminal relaxation time should be much longer than the complexation time, since an HMHEC chain contains more than two hydrophobes that must become unstuck during the terminal relaxation process of a chain. The trends in the mechanical spectra were the same for both the investigated AM concentrations. Figure 7 shows G′ at a single frequency (1 rads-1) for both 0.25 and 0.1 wt % AM with increasing M-R-CD concentration. At all M-R-CD concentrations, the mixtures with 0.25 wt % AM had a higher G′ than mixtures with 0.1 wt % AM, in line with the previous findings that an HMHEC-AM mixture forms the strongest gel with 0.25 wt % AM.17 The figure also includes G′ for increasing β-CD content in mixtures with 0.25 wt % AM. The decrease in G′ for mixtures with β-CD is slower than for mixtures with M-R-CD. This may be due to a poorer fit between the HMHEC hydrophobes and the β-CD than with the hydrophobes and M-R-CD.28 At 3.3 mM M-R-CD, the highest M-R-CD concentration in Figure 7, there are still effects of the AM in the samples. The viscosity at the same M-R-CD concentration in the AM free mixtures has already leveled off. The observed difference between the samples with and without AM is consistent with a competition between the AM and the CD molecules to form complexes with the HMHEC hydrophobes, that is, that AM to some extent prevents the CD complexation. Flow measurements could be performed at higher M-R-CD concentrations, and Figure 8 shows that the effect of AM is
Figure 10. Partial 1H NMR spectra of 0.25 wt % AM, 1.0 wt % HMHEC, and M-R-CD from bottom to top: 0.11, 1.1, and 9.9 mM M-R-CD. Well-resolved peaks are methyl (HMHEC hydrophobes and butanol) around 0.80 ppm, intermediate methylene groups (HMHEC hydrophobes) and C-3 (butanol) around 1.25 ppm, and C-2 (butanol) around 1.43 ppm.
detectable also at the highest M-R-CD concentration (10 mM) investigated. Diffusion. The self-diffusion of HMHEC and of AM in mixtures with AM is shown in Figure 9, where the HMHEC diffusion in the mixtures without AM is also included for comparison. The signal from M-R-CD was, unfortunately, disturbed by the AM and no quantitatively reliable results could be obtained from this component. The self-diffusion of both components of HMHEC and of AM are seen to increase with increasing concentration of M-R-CD. It is notable that none of these diffusion coefficients have leveled off even at 7 mM cyclodextrin and that the HMHEC diffusion is always much slower in the mixtures with AM. These results imply that there is still cross-linking by AM of the HMHEC molecules in the mixtures, also at the highest M-R-CD concentration. This is consistent with the viscosity data (Figure 8), which also show that there is an effect of AM at high M-R-CD concentrations. NMR Spectroscopy. The NMR spectra have been analyzed also in the mixtures with AM, Figure 10. In accordance with an earlier study,17 the intensity from the HMHEC hydrophobes disappears as the hydrophobes are complexed with AM because of a very large broadening of the peaks as a result of the slow dynamics of hydrophobes bound in quasi-immobile complexes with AM. Thus, in contrast to the experiments with CD, the spectra of hydrophobes complexed with AM are not accessible in high-resolution NMR experiments. Unfortunately, the AM samples also contain some butanol, which was not totally removed
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However, owing to the uncertainties in the relative intensities obtained by subtraction of the butanol peaks (presumably, these uncertainties are responsible for the relatively large scatter in the data), the data are not considered to be sufficiently reliable for a quantitative analysis in terms of a binding constant.
Concluding Discussion
Figure 11. Intensities of the HMHEC hydrophobe relative to the C-2 butanol peak; intermediate methylene (circles) and methyl (squares) groups with increasing M-R-CD concentration.
by the heat treatment (see the Experimental Section). The methyl and the C-3 methylene peaks from butanol overlap with the peaks from the polymer hydrophobes, but the C-2 methylene protons in butanol at 1.43 ppm do not overlap with any HMHEC hydrophobe peaks, and this can be used to estimate the butanol content. By comparing the intensity of the C-2 methylene peak from butanol with the M-R-CD peak at 5.1 ppm (not shown), the butanol concentration in the sample could be estimated. An independent estimate of the butanol concentration was also obtained by investigating the intensity of the C-2 butanol peak by adding known concentrations of butanol to an HMHEC-AM gel. Both experiments indicated a butanol content about 0.1 mM. The butanol in the samples can also form inclusion complexes, both with AM and with M-R-CD. The major part of the complexes in the mixture should, however, involve the HMHEC hydrophobes since the hydrophobe concentration is higher than the butanol concentration and the complexation constant for R-CD and butanol is quite low (0.092 mM-1).48 However, Figure 10 shows that the resonance peak from butanol also is shifted with increasing M-RCD concentration, indicating that there is a significant butanol complexation at high cyclodextrin concentrations. Because of the broad hydrophobe peaks and the overlap between the butanol and the HMHEC hydrophobe peaks, a chemical shift investigation of the HMHEC hydrophobes could not be performed. Instead, the changes in peak intensities could be studied by subtracting the butanol intensities. The intensity from the C-2 protons in butanol is the same as the intensity from the C-3 protons at 1.3 ppm, and the butanol contribution at that peak can be subtracted. The methyl peak at 0.8 ppm was treated in the same way, taking into account that the intensity of the methyl signal in butanol is 3/2 times higher than the intensity of the methylene peak. The result for increasing M-R-CD concentration is shown in Figure 11. Because of the much narrower lines of the HMHEC hydrophobes complexed with M-R-CD, compared to the AM complexes, the intensities from the HMHEC hydrophobes will increase when the AM complexes are replaced by CD complexes. The relative intensities for both the methylene and the methyl peaks increase fast at low M-R-CD concentrations and level off at higher M-R-CD concentrations, thus providing direct spectroscopic evidence of a change in complexation of the hydrophobes. (48) Ohtsuki, H.; Kamei, K.; Nagata, T.; Yamamoto, T.; Matsui, Y. J. Inclusion Phenom. Macrocyclic Chem. 2004, 50, 25.
This investigation has shown, as expected, that CD molecules bind to the HMHEC hydrophobes. To estimate the complexation constants, viscosity and chemical shift data were fitted to models assuming 1:1 complexation only. Models allowing additional complexation were found to contain too many parameters to be meaningful, but 2:1 complexes have been shown to occur in studies of other systems of comparable hydrophobic chain lengths.34,35 The apparent complexation constants obtained in our analysis are of a magnitude intermediate between the 1:1 and 2:1 complexation constants obtained for comparable mixtures involving surfactants with the same alkyl chain length as the hydrophobes of HMHEC. The low complexation constants obtained in our analysis are therefore most likely apparent complexation constants because of the influence of both 1:1 and 2:1 (CD:HMHEC hydrophobes) complexation. The existence of 2:1 complexes is further supported by self-diffusion data, which show that the concentration of bound M-R-CD molecules is about twice the concentration of HMHEC hydrophobes, and by the nonmonotonic variation of the NMR line shape from the methyl groups of the hydrophobes when CD is added. The AM/HMHEC complexation can be hindered by addition of CD to the cold mixture. Both NMR and viscosity data show that AM competes with the CD for formation of inclusion complexes with the HMHEC hydrophobes. The effect of AM decreases as the CD concentration increases in the mixture, but the CD molecules have not replaced all AM helices at the HMHEC hydrophobes even at the highest (10 mM) investigated M-R-CD concentration. This is evidenced by the observation that neither the self-diffusion of HMHEC or AM nor the viscosity of the mixtures has leveled off at the highest M-R-CD concentration. The AM/HMHEC complexation can also be hindered by the addition of, for example, surfactants, which compete with the polymer hydrophobes for the formation of complexes with AM. A previous study has shown that about 6 mM sodium dodecyl sulfate was need to completely eliminate all AM/HMHEC interactions.16 The CD concentration needed for total disappearance of the AM/HMHEC interactions is in the same order, but viscosity measurements show that there is a slight effect of AM in the mixture also at 10 mM, which is the highest CD concentration in the investigation. Acknowledgment. The authors thank Prof. Olle So¨derman, Markus Nilsson, and Geraldine Lafitte for valuable help with the NMR experiments and interpretations of the results. Dr. Krister Thuresson is acknowledged for useful discussions. We thank Dr. Karl-Erik Bergquist for performing the 1H NMR experiments. This work was financed by the Centre for Amphiphilic Polymers from Renewable Resources (CAP) (M. K.) and by the Swedish Research Council (L. P.). LA052177I
