Hydrophobically Modified Xanthan: An Amphiphilic but Not

Article pubs.acs.org/Biomac

Hydrophobically Modified Xanthan: An Amphiphilic but Not Associative Polymer Audrey Roy,† Sébastien Comesse,† Michel Grisel,† Nicolas Hucher,† Zied Souguir,‡,§ and Frédéric Renou*,† †

University of Le Havre, URCOM, EA 3221, FR CNRS 3038, 25 rue Philippe Lebon, B.P. 540, 76058 Le Havre Cedex, France Laboratoire Polymeres Biopolymeres Surfaces, University of Rouen, F-76821 Mont St. Aignan, France

‡

ABSTRACT: Hydrophobic octyl moieties have been grafted in various densities onto the carboxylic acid functions of xanthan under its ordered conformation. The outcoming amphiphilic and associative properties were studied by fluorescence spectroscopy and rheology. Results showed that the conformation of xanthan is not affected by the chemical modification and remains the same as the native one. Additionally, xanthan derivatives do not show any viscoelastic enhancement; nevertheless, their dynamics is strongly slowed down: the higher the grafting density, the slower the relaxation. We proved that hydrophobically modified xanthan, even being amphiphilic, does not exhibit any additional associating properties compared to the unmodified xanthan. The high stiffness of xanthan helices does not allow the derivatives to adopt the organization usually observed for flexible amphiphilic polymers. On the basis of these observations, a model depicting such a singular behavior is proposed.

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INTRODUCTION During the last three decades, water-soluble hydrophobically modified polymers have been intensively studied due to their many applications as thickeners or viscosity modifiers in various fields such as cosmetics, drug delivery, or oil recovery.1−3 Those specific properties result in their capability to self-associate in aqueous solution through intra-and/or intermolecular interactions between the hydrophobic moieties. According to their structure, associative polymers can be telechelic polymers defined as macromolecules containing one or two associating blocks at the end of the chain4−6and comb-like copolymers with a hydrophilic backbone bearing more or less randomly grafted hydrophobic moieties, often obtained by chemical modification of a water-soluble polymer, such as polysaccharides. Indeed, the latter class of associative polymers is of growing interest due to abundant raw materials, low cost, biocompatibility for medical applications, and biodegradability. Many studies regarding the chemical modification of cellulose,7,8 chitosan,9,10 pullulan,11,12 or alginate13,14 are available in the literature. Yet most of the polysaccharides subjected to such modifications can be considered as “model” polymers, i.e., they are characterized by a “simple” chemical composition, own a linear structure, and adopt a coil conformation in solution. Very few studies deal with the chemical modification of more complex polysaccharides, such as xanthan. Xanthan gum is an anionic polysaccharide produced by fermentation from Xanthomonas campestris. Its primary structure consists of repeated pentasaccharide units composed of a 1−4-linked-β-(D)-glucose backbone substituted on every © 2014 American Chemical Society

second unit with a charged trisaccharide side chain containing a acid between two D-mannoses. The inner and terminal mannoses can be, respectively, substituted by an acetate and a pyruvate group.15,16 The degree of substitution of both acetate and pyruvate can vary according to the X. campestris strain used and fermentation conditions.17−19 Xanthan also possesses carboxylic functions, located on the pyruvate and glucuronic acid of the side chain, thus making it possible to chemically modify xanthan in a controlled and specific manner. In aqueous solution, xanthan adopts two different conformations: an ordered, rigid double-helix strand structure at low temperature and high ionic strength, and a disordered, flexible coil state at high temperature and low ionic strength.20−22 This reversible order-to-disorder transition is characterized by a specific temperature named conformational temperature (Tm) which leads to a strong decrease in xanthan rheological properties.23 Tm depends on several parameters such as the acetate and pyruvate rates,24,25 the ionic strength,26,27 and/or the pH28 of the solution, thus making possible to control its conformation and properties in solution by varying these parameters. Only a few examples of modified xanthan are described in the literature.29−31 However, in all cases, the authors aimed to synthesize xanthan derivatives or xanthan hydrogels for drugs delivery. To the best of our knowledge, Mendes et al.32 are the only ones who synthesized a hydrophobically modified xanthan D-glucuronic

Received: November 19, 2013 Revised: February 17, 2014 Published: February 18, 2014 1160

dx.doi.org/10.1021/bm4017034 | Biomacromolecules 2014, 15, 1160−1170

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Scheme 1. Grafting Reaction of Octylamine onto Xanthan

depolymerization have been done as well, and the same DS were obtained, but the spectra were ill defined. The grafting density was calculated as follows:

in order to obtain systems able to encapsulate cells. Furthermore, the rheological behavior of their derivatives in aqueous solution was not studied. Moreover, all of the chemical modifications described in these papers are carried out on the hydroxyl functions of xanthan via an esterification process, which does not allow high regioselectivity for such reactions and can lead to cross-linking between xanthan chains. One way to overcome this issue is to modify xanthan on its carboxylic functions through an amidation reaction. Bejenariu et al.33 managed to graft adipic acid dihydrazide onto the carboxylic functions of xanthan by carrying out a peptide coupling reaction, though to create a hydrogel suitable for drug delivery. Here, we present the synthesis of amphiphilic xanthan bearing hydrophobic alkyl chains onto the carboxylic functions via a peptidic reaction with variable grafting densities. The structure and rheological properties of these derivatives were also investigated using fluorescence and rheological analyses.

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%octyl =

noctyl n′RU

(1)

with noctyl corresponding to the number of moles of octylamine determined by comparing the integration of the methyl protons of grafted amide (0.88 ppm) and CH3COONa (1.90 ppm, internal reference). n′RU corresponds to the number of moles of modified repeating units of xanthan. n′RU was calculated by dividing the introduced mass of xanthan by its repeating unit molecular weight. M′RU was determined by incrementing calculations using eqs 1 and 2.

M′RU = MRU + %octyl × Moctyl − %octyl × M H20

(2)

In eq 2, MRU is the molecular mass of nonmodified xanthan and Moctyl the molecular mass of octylamine. MRU was determined by 1H NMR. Since each repeating unit of unmodified xanthan contains 1 glucuronic acid and 0.49 pyruvic acid, the maximum grafting density that could be obtained is 149%. Polarimetry. The Tm of xanthan was determined using polarimetry. Polymer solutions at 1 g/L were prepared by dissolving the required amount of powder in ultrapure water under stirring at room temperature overnight. Then, the pH and the ionic strength were, respectively, adjusted to 4.3 with 0.1 M HCl and/or 0.1 M NaOH and to 1 mS/cm with NaCl. Afterward, the solutions were gently centrifuged for 3 min in order to get rid of air bubbles. Optical rotation measurements were performed at 436 nm on a Perkin-Elmer 241 polarimeter using a 1 dm path length cell (1 mL). Data were measured every 5 °C from 25 to 80 °C (after 30 min of thermal equilibrium at each temperature). A sigmoid shape of the optical rotation versus temperature curve was obtained, and Tm was determined as the temperature corresponding to the inflection point.24 Rheological Measurements. Polymer solutions at 2 g/L were prepared as mentioned above. Oscillatory shear studies were carried out with a stress controlled rheometer HR2 (TA Instruments) with a Peltier temperature control device using concentric cylinder geometry (bob diameter, 27.98 mm; length, 42.11 mm; gap, 1.2 mm). Solvent evaporation was avoided by covering the geometry with a low viscosity silicon oil. The storage (G′) and loss (G′′) moduli were measured as a function of the frequency (from 0.1 to 10 rad/s) at different temperatures (from 5 to 70 or 80 °C in the case of X29C8) within the linear viscoelasticity domain. Fluorescence Spectroscopy. Fluorescence measurements were realized on a Varian Cary Eclipse spectrofluorimeter using 8-anilino1-naphthalenesulfonic acid (1.8-ANS) as a fluorescence probe. The excitation wavelength was 350 nm, and the emission was recorded from 380 to 690 nm. Polymer solutions ranging from 0.1g/L to 5g/L were prepared by dissolving at room temperature the required amount of powder in 20 mM sodium acetate buffer at pH 4.5 containing 1.8ANS at 2 × 10−5 M. The solutions were stirred overnight.

MATERIALS AND METHODS

Materials. Xanthan was kindly provided by Danisco (France). The substitution degrees of acetate and pyruvate determined by 1H-NMR were 0.87 and 0.49, respectively. Its moisture and protein contents were, respectively, 10.8% (determination by thermogravimetric analysis) and 0.1% (determination by the Bradford procedure). 2,2,3,3-d(4)-3-(Trimethylsilyl)propionic acid sodium salt (TSP), 1ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC), N-hydroxysuccinimide (NHS), sodium acetate, octylamine, and 8-anilino-1naphthalenesulfonic acid (1.8-ANS) were purchased, respectively, from Alfa Aesar, Acros Organics, Merck, and Sigma Aldrich. All reagents were used without further purification. Synthesis. The synthesis process is described in detail in the Results (see Chemical Modification of Xanthan) and Discussion sections. Characterization Methods. 1H NMR Analysis. To determine the grafting density of our samples, 300 MHz 1H NMR spectra were recorded at 80 °C, using a Bruker Avance 300 spectrometer. For all analyses, solutions of xanthan (modified and not) were prepared in D2O at 5g/L according to the following procedure. It was experimentally observed that the high viscosity of our derivatives in aqueous solutions affected the relaxation of the polymer’s protons, thus making difficult any quantitative analysis of their spectra. One way to overcome this issue is to decrease the viscosity of the samples. Thus, prior to the NMR analysis, chemical depolymerization using hydrogen peroxide in alkaline solution was carried out according to the procedure developed by Wu.34 NaOH (7.4 μL, 1 M) and 25 μL of H2O2 were added to 1.5 mL of a solution of xanthan at 6g/L in D2O. The solution was then heated at 80 °C under stirring for 1 h and afterward cooled down in an ice bath for 30 min to terminate the reaction. Then, a part of the solution was freezedried to obtain 5 mg of polymer. The resulting powder was finally dissolved at room temperature overnight in 1 mL of D2O containing 5 × 10−3 M of TSP and sodium acetate as chemical shift and internal quantification references, respectively. NMR measurements before 1161


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Figure 1. 1H NMR spectra of the precursor (top) and X17C8 (bottom) in D2O at 80 °C.

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RESULTS Chemical Modification of Xanthan. In order to confer amphiphilic properties to xanthan, octylamine was grafted onto the carboxylic functions of the polysaccharide under its helical conformation. This choice was motivated by the fact that amide functions are able to form hydrogen bonds as carboxylic acids do. Because the carboxylic functions play an important role on the helical conformation of xanthan molecules via hydrogen bonds,26 whatever the grafting density, the number of hydrogen bonds remains the same. Therefore, here only the presence of the alkyl chains may impact the xanthan properties. The synthesis was conducted via a carbodimiide-mediated peptidic coupling reaction in water at room temperature. Using carbodimiide in coupling reactions has been frequently reported in the literature,11,14,35−37 and the procedure described here is an adaptation of existing ones. The reaction

took place in two steps, as described in Scheme 1. During the reaction, all the reagents (EDAC, NHS, and octylamine) were added in the same stoichiometry with respect to the carboxylic functions (see Table 1). Prior to the synthesis, a stock solution of xanthan (0.5% w/v, 400 mL) was dialyzed against ultrapure water buffered at pH 3 with HCl for 24 h to fully protonate the carboxylic functions of the polymer. A xanthan aqueous solution (0.1% w/v, 300 mL) was then prepared by dilution of this stock solution and left under stirring at room temperature until complete homogenization. Then EDAC, a water-soluble carbodiimide, and NHS, a coreagent, were added to the medium to activate the carboxylic functions of xanthan. The pH was afterward adjusted to 4.5 with 1 M HCl, and the solution was left under stirring at room temperature for 2 h. The acidic conditions promote the 1162


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protonation of EDAC’s nitrogen atoms,35 thus facilitating the coupling reaction. The second step consisted of the addition of an aqueous solution of octylamine to the medium, followed by an adjustment of the pH to 10 with NaOH, in order to form the desired amide through the nucleophilic attack of octylamine onto the activated carboxylic functions of xanthan (Scheme 1). The reaction was stirred for 16 h at room temperature. Several experiments realized at different pH (4.5, 7, and 10) for this step showed that the reaction was much more efficient at pH 10, whereas it failed in acidic conditions. These results can be explained by the basic properties of octylamine (pKa = 10.65). Indeed, at pH 4.5, octylamine’s nitrogen atom is mostly protonated. As a consequence, the nucleophilic behavior fails, and no reaction occurs with the activated carboxylic functions. On the contrary, at pH 10, the nucleophilic properties of octylamine are preserved, thus favoring the reaction. Purification of the polysaccharides was conducted by successive dialysis steps against HCl (pH 3 for 8 h), 0.1 M NaCl (24 h), and distilled and ultrapure water during 8 days. The helicoidal conformation adopted by xanthan was checked at each step of this synthesis by polarimetry. In both cases, a sigmoid shape of optical rotation versus temperature curve was obtained, which is characteristic of an order-todisorder transition (data not shown) from a rigid, helix strand structure at low temperature to a disordered coil at high temperature, the transition temperature being about 65 °C. One may presume a destabilization of such a structure during the modification due to the hydrophobic moieties. Nevertheless, on the one hand, we replaced the carboxylic functions by amide functions in order to minimize the impact of hydrogen bonds (as mentioned above). On the other hand, the measurements showed that the modification conditions do not seem to destabilize much the helical structure of xanthan. Indeed, since the synthesis was carried out at room temperature, we can conclude that xanthan remains under its ordered, helical form during the grafting process. Precursor refers to a xanthan that underwent all of the above synthesis and purification procedures but without the addition of the coupling agents during the first step of the modification process in order not to graft octylamine. Therefore, the precursor is our reference as an unmodified xanthan. The nomenclature used for the modified polysaccharides, also called derivatives, is XAC8, where A is the grafting density (i.e., the number of octylamine chains per xanthan repeating unit), and C8 stands for the number of carbon atoms in the alkyl chain. The grafting density of the different modified polymers was quantified by 1H NMR at 80 °C in the presence of two internal references, CH3COONa (peak at 1.90 ppm) and TSP (peak at 0 ppm). Figure 1 represents the NMR spectra of the precursor (top) and X17C8 (bottom). For the precursor, the characteristic spectrum of xanthan is obtained, with two peaks at 1.45 ppm and 2.16 ppm attributed to pyruvate and acetate groups,38 respectively. No peak corresponding to octylamine can be observed on this spectrum. Bearing in mind that octylamine was added to the precursor during the second step of the synthesis, we point out that this spectrum proves that the whole purification procedure is efficient to remove all ungrafted octylamine. For X17C8, additional peaks at 0.98, 1.29, and 1.69 ppm can be observed. They are attributed to the grafted octyl chain (CH2 protons of the octyl chain at 1.29 and 1.69 ppm, and methyl proton of octylamide at 0.89 ppm). We also notice a

split of the pyruvate peak into two signals, one corresponding to pyruvate groups bearing a carboxylic function (1.47 ppm, as precursor) and the second (at 1.51 ppm) corresponding to pyruvate groups bearing an amide function as the result of the chemical modification. By integrating the peak at 1.51 ppm, one can roughly estimate the proportions of modified pyruvate and glucuronic acid. Results show that approximately half of the modified carboxylic functions are located on the pyruvate. Knowing that the xanthan used owns half pyruvate compared to glucuronic acid function, one can conclude that pyruvic acids are twice more reactive than the glucuronic acids. This could be explained by the better accessibility of the pyruvate groups compared to glucuronic ones since the former are located at the extremity of the side chain. The integration of the methyl peak of octyl group (at 0.89 ppm) and CH3COONa (internal reference, at 1.90 ppm) allowed the calculation of the grafting density of modified xanthan for each sample (see Materials and Methods for calculations). The grafting density is comprised between 0 and 29% according to the experimental conditions used during the synthesis process, which are summarized in Table 1. Table 1. Experimental Conditions of the Grafting Reaction of Octylamine on Xanthan and Grafting Density entry

XAC8

nEDAC/nCOOH

noctylamine/nCOOH

grafting density (%)

1 2 3 4 5

precursor X8C8 X13C8 X17C8 X29C8

0 2.2 2.7 2.7 3.5

3.5 2.2 2.7 2.7 3.5

0 8 13 17 29

Modified xanthans with varying grafting densities were obtained by changing the stoichiometry of EDAC, NHS, and octylamine with respect to carboxylic functions of xanthan. Entries 3 and 4 show that under the same reaction conditions the grafting densities are within the same range. This low discrepancy can be explained by some experimental differences such as small temperature variations and the inherent difficulty related to the modification of such a complex polysaccharide. However, the synthesis conditions authorize the control of the grafting density of our polymers. Moreover, with the process developed here, it is possible to obtain xanthan derivatives with high grafting densities but still soluble in water. Hydrophobic Interactions. ANS is a well-known fluorescence probe used in biochemistry to study the local polarity and organization of biological systems such as proteins or membranes.39−42 Indeed, its emission spectrum is characterized by a large blue shift when the probe is displaced from a high polar to a nonpolar medium.43 Moreover, ANS is nonfluorescent in the presence of water, whereas it is highly fluorescent in nonaqueous solutions.44,45 Such properties explain why ANS is also used to detect the presence of hydrophobic microdomains resulting from intra-and/or intermolecular interactions for associating polymers.46,47 Figure 2a shows the fluorescence spectra of ANS for the precursor and X29C8 at different concentrations. For clarity, the other polysaccharide spectra were not plotted, yet all of them exhibit the same features as those presented. In Figure 2a, we can notice an increase of the fluorescence intensity for both xanthans when polymer concentration increases. This phenomenon is more pronounced for X29C8 than for the precursor. This increase of fluorescence is 1163


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Figure 2. (a) Fluorescence emission spectra of ANS in the presence of X29C8 (solid lines) or the precursor (dashed lines) at different concentrations. (b) Evolution of the maximal emission wavelength of ANS as a function of the polymer concentration for modified xanthans and the precursor.

Figure 3. (a) Maximal fluorescence intensity of ANS versus polymer concentration for modified xanthans and the precursor. (b) Evolution of the fluorescence intensity as a function of the normalized concentration for modified xanthans and the precursor. The symbols are the same as those in panel a.

All samples present a critical concentration, called Cc, from which the intensity noticeably increases. This concentration is defined as the onset of the upward shift of the curve. We can also notice that this concentration slightly decreases from 0.8g/ L for the precursor to 0.4g/L for the most grafted sample. This range of concentration is in agreement with the one corresponding to the sharp decrease of the maximum wavelength, as illustrated in Figure 2b. The results described here indicate that, above Cc, there is migration of water molecules away from the vicinity of ANS molecules, and this phenomenon is enhanced when the grafting density increases. This confirms that above a critical concentration, ANS detects the presence of apolar regions resulting from interactions between xanthan chains. Before explaining the organization of derivatives in solution above Cc, it is important to address the issue of the precursor. It is well-known that hydrogen bonding is of primary importance for water-soluble polymer properties in aqueous media.26

accompanied by a shift toward blue wavelength of the emission of the probe with the concentration in both cases. Figure 2b represents the evolution of the maximum emission wavelength with the concentration for all polysaccharides. The curves are almost identical for all samples with a sharp decrease of the maximum wavelength from 520 to 530 nm to 465−470 nm between 0 and 1g/L. Above this concentration, the maximum wavelength does not evolve much with concentration and tends to stabilize at 465 nm. In water, ANS emits at 520 nm, whereas in a hydrophobic environment, the maximum emission is observed between 460 and 480 nm.46,48 These results clearly indicate that ANS molecules are surrounded by an increasingly apolar medium when the polymer concentration rises, whatever the sample. Therefore, it appears that all xanthans, even the precursor, form hydrophobic regions able to interact with ANS beyond a critical concentration. Figure 3a shows the evolution of the maximal fluorescence intensity with polymer concentration for all samples. 1164


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Figure 4. (a) Frequency dependence of the storage G′ (close symbols) and loss G′′ (open symbols) shear moduli at different temperatures for the precursor at 2g/L, pH 4.3, and IS = 1 mS/cm. (b) Frequency−temperature master curve of the frequency dependence of the storage and loss modulus at Tref = 20 °C for the precursor at 2g/L.

above Cc. These curves have been normalized by C/Cc, and the results are plotted in Figure 3b. All curves superimpose very well and give the same curve as the precursor. This suggests that ANS behavior is identical whether the probe is in the presence of derivatives or the precursor, but only shifted toward lower concentrations when the grafting density increases. Thus, we can conclude that all grafted xanthans adopt a similar organization in solution as the precursor above Cc, and that Cc most likely corresponds to C**. It is well-known that grafting hydrophobic moieties to a hydrophilic polymer confers to the latter an attractive potential that decreases C**,55,56 which explains the observed results. Nevertheless, the influence of the grafting density on Cc appears quite low, which implies that this mechanism is notably moderate. To conclude, fluorescence measurements proved that unmodified xanthan possesses very weak associating properties which are due to the formation of intermolecular H-bonds above a critical concentration. Additionally, we demonstrated that grafting hydrophobic moieties onto its backbone does not modify significantly its associating character. Thus, the whole observations suggest that the grafting density does not affect much the structure and organization of xanthan chains in solution. Rheological Properties. Before characterizing hydrophobically modified xanthans solutions, we studied the viscoelastic properties of the precursor over a wide range of temperature. Figure 4a shows the frequency dependence of G′ and G′′ for this sample at 2g/L at 5, 20, 40, and 60 °C. For clarity, the other temperatures were not reported on this graph. The precursor exhibits the typical features of a xanthan solution at such a concentration.57 Indeed, at 5 °C, for ω ≥ 0.37 rad/s, the precursor displays a weak gel behavior due to intermolecular H-bonding, with G′ higher than G′′. The two moduli cross at ωref = 0.37 rad/s, corresponding to the relaxation frequency. This frequency shifts toward higher values when the temperature increases and is out of the explored frequency range up to 60 °C. For this temperature, G′′ is higher than G′ on the whole range of frequencies investigated, which is typical of a viscoelastic liquid. With these data, it is possible to construct a master curve by means of frequency−temperature superposition, as described in the literature for xanthan.57−59 The resulting master curve for the precursor is plotted in Figure 4b and shows that data superimpose very well on the whole range of temperatures and frequencies investigated. At high frequencies and low temper-

Indeed, intramolecular H-bonding is at the origin of the helical conformation of xanthan once the electrostatic repulsions are screened (at high ionic strength), while intermolecular Hbonding is suspected to be the most important interchain interactions responsible for its weak gel properties. According to the fluorescence results presented, this polymer has hydrophobic regions when its concentration is above Cc = 0.8g/L, even though this polysaccharide does not bear hydrophobic grafted moieties (as evidenced by its 1H NMR; see Figure 1). Xanthan is a hydrophilic polymer; nevertheless, this polysaccharide can retain hydrophobic compounds via its apolar cellulosic backbone, as demonstrated by aroma retention experiments.49,50 Therefore, it is most likely that the increase in ANS fluorescence measured can be explained by similar interactions. Furthermore, we noticed that Cc is very close to the entanglement concentration C** value usually found in the literature for xanthan in aqueous solution.51,52 C** corresponds to the creation of a viscoelastic network of xanthan chains through interchain H-bonding interactions, thus explaining why this phenomenon is so critical. The formation of such a network when C becomes higher than C** would promote the expulsion of water molecules from the vicinity of ANS molecules. For the modified xanthans, one hypothesis is that these polysaccharides adopt, above Cc, a conformation in solution similar to flexible amphiphilic polymers. Indeed, the latter are known to form well-defined hydrophobic microdomains in solution above the critical aggregation concentration CAC, as molecular surfactants do.53 However, in our case, this behavior seems most unlikely for two reasons. First, we demonstrated that, in our experimental conditions, xanthan chains remain in helical, very stiff ordered conformation, characterized by a persistent length of 120 nm.54 Hence, it is impossible for the chains to fold themselves in order to form such microdomains. Furthermore, the precursor itself presents the same fluorescence features as the derivatives, although this xanthan is not modified. All of these suggest that modified xanthans cannot adopt the same organization in aqueous solution as the one usually observed for flexible associative polysaccharides. In Figure 3a, we can observe the very close similarities between the fluorescence curves of modified xanthans and the one of the precursor, the only difference being the critical concentration, which decreases when the grafting density rises, resulting in an increase of the intensity at a given concentration 1165


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atures, the precursor exhibits a weak gel behavior, with G′ higher than G′′ and both moduli weakly frequency-dependent, with G′ ∼ ω0.45 and G′′ ∼ ω0.3. On the other hand, at low frequencies and high temperatures, the system exhibits a liquidlike behavior, typical of xanthan, with a higher frequency dependence for both moduli: G′ ∼ ω1.8 and G′′ ∼ ω0.94, close to the frequency dependence of a purely Maxwell liquid. The same approach was applied to each modified xanthan. Figure 5 represents the resulting master curves for X8C8, X29C8, and the precursor at 2g/L (for clarity, the other curves are not shown).

Figure 6. Evolution of the relaxation time as a function of the grafting density for modified xanthans and precursor.

they strongly impact the rheological properties of this polysaccharide at low frequencies. Moreover, this impact is tunable with the grafting density. Figure 7 shows the horizontal shift factors (aT) used for constructing the different master curves as a function of the inverse absolute temperature. We can notice that log(aT) evolves linearly with the inverse absolute temperature for all derivatives and xanthan up to 60 °C. Beyond this limit, we observe a strong decrease of the value of aT of about one decade due to the order-to-disorder transition of the xanthan chains (Tm = 65 °C). Thus, those values were not taken into account for the calculation of the activation energy. For all samples, values of aT spread over about three decades, in agreement with the literature.57,59 Hence, the temperature dependence of the viscoelastic properties of all xanthans can be described by Arrhenius’ law, i.e., defined by a unique activation energy. The activation energies of derivatives are slightly higher than that of the precursor, yet no correlation can be found between the values of Ea and the grafting density. Moreover, these activation energies are comprised between 60 and 100 kJ/mol, which is within the order usually reported for unmodified xanthans,57,59 and thus indicating that the grafting density barely affects Ea. The similar Ea values for both the precursor and derivatives suggest that the contribution of the alkyl chains to the temperature dependence of the viscoelastic properties is minor compared to that of the H-bonding contribution. Therefore, we can conclude that the temperature dependence of the rheological properties is somehow universal for all xanthans, whatever the grafting density. Such results point out that the relaxation processes involved in modified xanthans are similar to the ones of nonmodified xanthan, thus confirming that grafted xanthans adopt the same organization as the precursor. The presence of grafted moieties also affects the flow behavior of xanthan in solution. Figure 8 shows the master curves of the dynamic viscosity (η′) for all samples. The Cox− Merz rule for xanthan has been reported by several authors59,61 within the Newtonian plateau and up to 4 decades of shear rate beyond the linear regime, while at higher shear rate, a small discrepancy is observed between shear viscosity and dynamic viscosity. Nevertheless, results are still comparable. At high shear rates, all polysaccharides show the same shearthinning behavior, whatever the grafting density. Thus, the presence of hydrophobic moieties does not impact the flow properties of xanthan in this range of frequency (shear rate).

Figure 5. Frequency−temperature master curves of G′ (filled symbols) and G′′ (open symbols) for X8C8, X29C8, and the precursor at 2g/L (Tref = 20 °C).

Frequency−temperature superposition also gave very good results for all the derivatives. At high frequencies, all of the modified xanthans have a weak gel behavior similar to that of the precursor. We can also notice that the storage modulus very weakly increases with the grafting density, whereas the loss modulus remains constant. These observations seem to indicate that the number of physical elastically active chains (cross-link density) between the precursor and the most grafted xanthan is almost identical. This result confirms that all samples have the same organization in solution and that the grafted moieties do not affect much the rheological properties of xanthan within this frequency range, in agreement with the fluorescence results. At low frequencies, however, the behavior of derivatives is clearly different from the precursor. We can observe a decrease of the frequency dependence of both moduli with the grafting density, accompanied with a strong decrease in the relaxation frequency, hence showing a very pronounced slowing down of the chains dynamics. The dynamics of a system can be characterized by its relaxation time τ = 1/ωref. The evolution of the relaxation time with the grafting density of our systems is presented in Figure 6. More precisely, the relaxation time follows a power law with an exponent of 5.5. It indicates that the relaxation of xanthan derivatives strongly depends on the grafting density. X29C8 is thus characterized by a relaxation time about 30 000 times higher than that for the precursor. This scaling law allows us to predict the dynamics of xanthan in this range of grafting density. To explain these results, we propose that our system undergoes sticky relaxation dynamics60 due to intermolecular hydrophobic interactions between the grafted moieties within the concentrated regime. So even though grafted moieties do not influence the organization of xanthan chains in solution, 1166


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Figure 7. Horizontal shift factors (aT) as a function of the inverse absolute temperature. Solid lines represent linear regressions; table, activation energy (Ea) for each xanthan.

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DISCUSSION

The organization of a series of hydrophobically modified xanthans in aqueous solution was investigated by ANS fluorescence analyses. Results showed that each polysaccharide can be characterized by a critical concentration, corresponding to the entanglement concentration C**, from which there is an increase of the fluorescence intensity, which means that all polymers form hydrophobic regions able to interact with the ANS probe when the concentration rises. Moreover, this critical concentration decreases when the grafting density increases. By superimposing each fluorescence curve to each other after normalizing them by C/C**, it was possible to construct a single curve similar to that of the precursor (Figure 3b). This suggests that ANS behavior is identical whether the probe is in the presence of derivatives or the precursor but that it only shifted toward slightly lower concentrations when the grafting density increases. In other words, all xanthan derivatives adopt the same organization in aqueous solution as the precursor, whatever the grafting density. Thus, chemical grafting does not modify the structure of xanthan. Characterization of the viscoelastic properties of the different systems gave further evidence supporting this hypothesis. First, comparison of the master curves obtained for each sample showed that all of them have the same weak gel behavior as the precursor at high frequencies, whatever the grafting density. In particular, the very weak increase in the storage modulus with the grafting density indicates that all xanthans have approximately the same number of physical elastically active cross-links. This result implies that the presence of hydrophobic moieties does not modify much the xanthan network and, thus, the organization of its chains in solution. Moreover, the temperature dependence of the rheological properties is universal for all xanthans and characterized by a unique activation energy whose values are slightly different from the ones for a nonmodified xanthan. Therefore, grafted xanthans present the same relaxation mechanism as the precursor, thus corroborating our proposed hypothesis. However, the evolution

Figure 8. Master curves of the dynamic viscosity (η′) as a function of ω·aT with Tref = 20 °C at 2g/L.

However, at low frequencies, the precursor presents a Newtonian plateau, with η0 = 2 Pa·s for ω < 0.01 rad/s, whereas xanthan derivatives still exhibit a shear-thinning behavior. Hence, the shear-thinning behavior is more and more pronounced as the grafting density increases and spans over a larger frequency range. For example, at 10−5 rad/s, X29C8 shows a viscosity 250 times higher than that for the precursor. Therefore, the grafted moieties strongly influence the flow behavior of xanthan at low frequencies. Indeed, the intermolecular hydrophobic interactions developed between these moieties considerably strengthen the suspending ability of xanthan at rest (or under very low shear), whereas the flow properties remain identical for all xanthans, whatever their grafting density, at high shear rates. This ability is reinforced when the grafting density increases. 1167


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Scheme 2. Schematic Representation of the Precursor and Grafted Xanthan in Aqueous Solution above C**

conformation. In the present work, we demonstrate that grafting hydrophobic moieties to xanthan create hydrophobic intermolecular interactions with no xanthan conformation modification, nor its associative character; surprisingly, only the overall relaxation is strongly affected by the presence of such moieties. Here, the high rigidity of the xanthan helical structure does not allow the chains to fold themselves to create such hydrophobic microdomains. Indeed, here we show that an important change in rheological properties can be obtained without any change in polymer conformation. The opposite behavior was also reported; indeed, ionic amphiphilic block copolymers are universally nonsurface active since they are not absorbed at the air/water interface but form micelles in solution when the required conditions are satisfied, whether the hydrophilic block is anionic62,63 or cationic.64 The lack of surface activity of these polymers is attributed to the image charge−effect at the air/water interface. In those cases, hydrophobic modification promotes associativity without any interface properties modification. The latter examples and the present study demonstrate that for modified polymers, conformation and macroscopic properties are not systematically correlated. In addition, Mendes et al.32 grafted hydrophobic palmitoyl groups to xanthan under its helical conformation and found that the obtained amphiphilic polysaccharide is able to build a multilayered lamellar structure. To explain these results, they proposed a model where the grafted moieties are located on the side chain of xanthan, therefore at the surface of the helix, and can interact with each other through intermolecular hydrophobic interactions to form the double layer of helices. Their findings are in agreement with the model proposed in the present work.

of the relaxation time with the grafting density follows a scaling law with an exponent of 5.5, which highlights the very strong impact of the grafting density on the relaxation of xanthan. Recently, Choppe et al.59 demonstrated that the rheological properties of xanthan cannot be explained by the reptation relaxation of entangled polymers. They proposed that the viscoelasticity is linked to the formation of a transient network of helices cross-linked by bonds with a finite lifetime. On the basis of that hypothesis, for the derivatives, hydrophobic interactions developed between the grafted alkyl chains increase the lifetime of such bonds but do not modify their number since the values of both moduli are not affected by grafting and thus do not contribute to the elasticity. To summarize, the presence of hydrophobic moieties does not modify the xanthan conformation in water; in addition, their contribution to viscoelasticity is negligible compared to that of the H-bonds network; nevertheless, they strongly affect the overall relaxation. To depict such a behavior, we propose a model which describes the organization and the dynamics of the unmodified xanthan and its derivatives (see Scheme 2 left and right, respectively). The derivatives adopt exactly the same organization as the precursor, but the intermolecular hydrophobic interactions strongly affect the transient network lifetime. Therefore, the rheological behavior of the grafted xanthans would be explained by an additional sticky relaxation of the transient network due to intermolecular hydrophobic interactions. The corresponding dynamics is governed by the same relaxation processes than the precursor but only strongly impeded by the hydrophobic moieties. Hence, we can conclude that the hydrophobically modified xanthans do not possess any extra associating properties than their precursor, even though they have amphiphilic characters. Usually, hydrophobic modification of flexible polysaccharides leads to the formation of well-defined hydrophobic microdomains above a critical aggregation concentration (CAC) in aqueous solutions due to intra and/or intermolecular interactions.36,47 Those interactions induce the occurrence of specific molecule conformation and organization, which is at the origin of the specific rheological or interfacial properties observed for such modified polysaccharides.8,14 Thus, the macroscopic properties remain finely related to the polymer

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CONCLUSIONS Hydrophobically modified xanthan with tunable grafting density comprised between 0 and 29% was obtained by chemical grafting of octylamine onto the carboxylic functions of xanthan chains using a two-step carbodimiide-mediated coupling reaction in water. Under these conditions, xanthan chains remain in their ordered, rigid helix conformation during the entire modification process. 1168


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In the present study, grafting hydrophobic alkyl chains onto xanthan molecules create additional intermolecular interactions but without modification of either the conformation of the chains or of their associating properties. Furthermore, the viscoelastic properties remain unchanged: only the dynamics of the network is strongly slowed down; the more grafted, the slower is the relaxation. Hence, the original conformational and rheological characteristics of modified xanthans are contrary to what is usually observed for associating amphiphilic polymers, for which the chains adopt a specific conformation due to the formation of more or less well-defined hydrophobic microdomains, which are at the origin of their peculiar rheological properties. Indeed, the high stiffness of xanthan helices does not allow the chains to form such hydrophobic microdomains in solution. Moreover, these hydrophobic interactions considerably strengthen the suspending capability of xanthan at rest but without modifying its shear-thinning behavior at high shear rates. This last property, tunable with the grafting density, can have potential applications in formulation as stabilizing and thickening agents. So far, this study has been conducted on a xanthan under its ordered, rigid conformation. It would be interesting to observe whether the same properties appear for a xanthan modified under its disordered conformation. The influence of the length of the hydrophobic moieties on those properties is also currently under investigation.

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AUTHOR INFORMATION

Corresponding Author

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

Z.S.: Celenys SAS, 75 route de Lyons la forêt, 76000 Rouen, France. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge Professor Luc Picton from PBS, Rouen (France), for fruitful discussions concerning the chemical modification. This work is supported by the Région Haute Normandie (Graduate Fellowship to A.R.).

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Hydrophobically Modified Xanthan: An Amphiphilic but Not

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