Scleroglucan Gelation by in Situ Neutralization of the Alkaline Solution

May 9, 2003 - Structure-Function Relationships in Glycopolymers: Effects of Residue Sequences, Duplex, and Triplex Organization. Marit Sletmoen , Bjø...
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Biomacromolecules 2003, 4, 914-921

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Scleroglucan Gelation by in Situ Neutralization of the Alkaline Solution Einar Aasprong,† Olav Smidsrød,† and Bjørn Torger Stokke*,‡ Norwegian Biopolymer Laboratory, Department of Biotechnology and Department of Physics, The Norwegian University of Science and Technology, NTNU, NO-7491 Trondheim, Norway Received December 20, 2002; Revised Manuscript Received April 14, 2003

Scleroglucan gels were prepared by neutralization of aqueous alkaline solution of scleroglucan by in situ release of acid. Transition of scleroglucan chains in a disordered state to the triple-helical state result in cross-linking of the polysaccharide. The pH and the storage and loss moduli, G′ and G′′, were determined as a function of time after initiating the pH reduction. Experiments were performed in the temperature range from 20 to 90 °C and in the polymer concentration range from Cp ) 10-200 mg/mL. The concentration dependence of the apparent plateau value of the storage modulus showed a lower critical concentration, Cp,0, in the range 12-15 mg/mL needed for gelation and a second power dependence of G′ on concentration in the range 50-200 mg/mL. In situ pH reduction was achieved using formamide (methanoic acid) or ethyl acetate (ethyl ethanoate) which hydrolyze in alkaline solution, yielding carboxylic acids. The more rapid hydrolyses of ethyl acetate compared to formamide yielded a faster decrease of pH in solution and a more rapid increase of the storage modulus. The present gelation process of scleroglucan based on the in situ reduction of pH has advantages in applications where external control of the conditions is difficult to achieve. 1. Introduction Gelation of scleroglucan and other comblike (1f6)-β-Dgluco-(1f3)-β-D-glucans can be achieved by formation of covalent or physical cross-links. Scleroglucan cross-linked by Schiff-base formation between aldehyde groups introduced selectively in a fraction of the scleroglucan side-chains, and primary amines with a functionality of at least two, is one type of covalently cross-linked scleroglucan gels. By using the polyfunctional primary amine, chitosan, as the cross-linker, one obtains an all-polysaccharide gel.1,2 Another example of covalently cross-linked scleroglucan is obtained by the use of diethyl squarate reacting with aminoalkylated scleroglucan.3 Physical gelation by interchain complexation can be achieved for scleroglucan by the use of zirconium4 or Borax5 and for carboxylated scleroglucan using Cr(III).6 By utilizing the triple-helical structural motif of scleroglucan in aqueous solution, physical gels can also be made. This requires a denaturation of the scleroglucan and then reconstituting the solvent conditions favoring the triple-helical state. Such a process closely resembles the regeneration of duplex or triplex segments in thermosetting gelation of gelatin.7 The conformational transition temperature for the melting of the triple-helical structure of high molecular weight scleroglucan or schizophyllan in aqueous solution is about 135 °C.8,9 Compared to gelatin (or collagen), this is beyond reach for making thermosetting gels without making use of a pressurized vessel. The triple-helical structure of * To whom correspondence should be addressed. Phone: +47 73 59 34 34. Fax: +47 73 59 77 10. E-mail: [email protected]. † Department of Biotechnology. ‡ Department of Physics.

scleroglucan is also reported to be destabilized in dimethyl sulfoxide or alkaline conditions.8 The curves in the conformational phase diagram, as a function of temperature and the OH- concentration, represents the midpoints of conformational transition, between the triple helical conformations I and II and between these and the dissociated state (Figure 1).10 Triple helix II prevails at moderate or high temperatures and neutral or acidic conditions. The transition from triple helix I to II depends strongly on solvent conditions.8,11,12 In particular, the finding that triple helix I at neutral pH is stabilized by replacement of water with D2O was used as a basis for suggesting changes in detailed localization of the side-chains being involved in this transition. The transition to triple helix I at temperatures below 5 °C is also reported to induce association between the triple helices resulting in the formation of a weak gel at sufficiently large concentrations.13,14 The temperature-induced melting of triple helix I to II at constant pH showed a reduction in the cooperative unit size with increasing pH, from 300 repeating units for pH below 10 to about 50 at pH 12.10 The solvent conditions can be changed to cross the strand-separation transition and thereby utilized for making physical scleroglucan gels. Formation of physical gels by (1f6)-β-D-gluco-(1f3)β-D-glucans with junction zones based on the triple-helical structural motif have been reported.9 Examples are cooling of schizophyllan solutions heated to 160 °C and dilution of schizophyllan dissolved in dimethyl sulfoxide by water to a water weight fraction favoring the triple-helical state relative to the single stranded state. Gels are formed in these latter examples if the polysaccharide concentration is above a gelation threshold. Similar to the macroscopic gels being

10.1021/bm025770c CCC: $25.00 © 2003 American Chemical Society Published on Web 05/09/2003

Scleroglucan Gelation by in Situ Neutralization

Biomacromolecules, Vol. 4, No. 4, 2003 915

in situ approach is also preferable due to reduced spatial variation in parameters used to induce gelation. 2. Experimental Section 2.1. Chemical Reactions and Equilibria. In the present study, gelation is induced by an in situ pH reduction brought about by the alkaline hydrolyzes of either formamide (eq 1a) or ethyl acetate (eq 1b). Formamide hydrolyses yielding methanoic acid and ammonia present in equilibria according to eqs 2a and 3. Ethyl acetate hydrolyses yielding ethanoic acid and ethanol. Ethanoic acid is present in equilibrium according to eq 2b. Alkaline Hydrolyzes of Formamide Figure 1. Conformational phase diagram for scleroglucan in alkaline solution.10 The dashed line illustrates the part of the phase diagram that has not been experimentally determined. The change in [OH-] for 50 mg/mL of scleroglucan in aqueous 0.316 M NaOH after adding formamide to a final concentration of 0.632 M is given at 20 °C (O) and 50 °C (0), obtained from the results reported in Figure 2. Symbols are plotted for each 1/2 hour after mixing, the arrows giving the direction of change.

formed from the two conditions presented above, pregel clusters have been reported to be formed upon neutralization of alkaline solutions of dispersed (1f6)-β-D-gluco-(1f3)β-D-glucans.10 The formation of a gel by these processes competes with regeneration of linear triple helical structures or formation of macrocyclic topologies.15,16 These types of species become less abundant the larger the concentration and chain length with the concomitant increased fraction of larger clusters.17 The physical gelation processes of (1f6)-β-D-gluco(1f3)-β-D-glucans by at least a partial regeneration of the triple-helical structure have so far been based on external control of the conditions inducing the gelation, e.g., by using a thermostated bath for controlling temperature. In the present work, we explore the possibility of using in situ reduction of the pH from an alkaline solution where the triple-helical structure is dissociated and thereby controlling a crucial parameter for the formation of a physical gel. Formamide or ethyl acetate is used as additives to aqueous, initially alkaline, solutions. Both formamide and ethyl acetate hydrolyze to carboxylic acids, reducing pH from about 13. The present gelation process of scleroglucan based on the in situ reduction of the pH has advantages in applications where external control of the conditions is difficult to achieve. Permeability modification of oil reservoirs is one such example. The purpose of a gel selectively placed in high permeability layers of the formation will be to divert subsequently injected fluid into formation zones of the oil reservoir with lower permeability. Such a treatment is envisaged to increase the sweep efficiency and delay the water breakthrough in the production well. This process requires that the gelation time can be controlled in a suitable range for the temperature profile of the reservoirs and that the gel stability is sufficient to yield an economic viable process. At the same time, gel degradation must be possible if the gel has not been correctly placed in the reservoir. The

HCONH2 + OH- f HCOO- + NH3 (v)

(1a)

HCOO- + H+ T HCOOH

(2a)

+

+

NH3 + H T NH4

(3)

Alkaline Hydrolyzes of Ethyl Ethanoate CH3COOC2H5 + OH- f CH3COO- + C2H5OH (1b) CH3COO- + H+ T CH3COOH

(2b)

The alkaline hydrolyzes of esters and amides have been reported18 to follow the second-order tetrahedral mechanism. In the first step, a nucleophilic attack by OH- at the carbonyl group gives a tetrahedral intermediate. In the second step, ammonia or ethanol respectively leaves the intermediate, thus leaving the methanoic or ethanoic acid. The rate constant of eq 1b is expected to be higher than that of 1a.18 For all practical purposes, the equilibria described in eqs 2a and 2b may in this study be considered totally shifted to the left as the pH during the gelation process is far on the alkaline side of the respective pKa values (3.74 and 4.76). In the case when formamide is used for in situ pH reduction, NH3 will be formed according to eq 1a in the same amount as OH- is consumed. Thus, as the pH is lowered in situ toward the pKa value of eq 3 (9.24), a sufficient amount of NH3 will be available to buffer the solution thus preventing further reduction of pH. Buffering by Dissolved Scleroglucan scleroglucan-O- + H+ T scleroglucan-OH

(4)

The deprotonation of the scleroglucan hydroxyl groups contributing to the destabilization of the triple helical structure also buffers the solution. Dissolving scleroglucan in alkaline solution will thus lower the pH by a magnitude that depends on the concentration of scleroglucan. During the gelation process induced by decreasing pH, the protonation of the hydroxyl groups are expected to reduce the rate of change in pH somewhat. The Ionic Product of Water H2O T OH- + H+

(5)

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In an alkaline solution, the temperature dependence of the ionic product of water yields a significant change in pH with temperature, [OH-] kept constant. A solution of 0.316 M NaOH being pH 13.5 at 25 °C will be pH 12.8 at 50 °C and pH 11.8 at 100 °C due to the temperature dependent increase in the ionic product of water. 2.2. Materials. A purified scleroglucan (Actigum CS-11, kindly provided by Sanofi Bioindustries, France) was used. The intrinsic viscosity [η] of this scleroglucan sample has been reported2 to 3000 mL/g in double distilled water and 3600 mL/g in 0.01 N NaOH at 20 °C and shear rate γ˘ ) 5 s-1. Using the experimentally determined relation between weight average molecular weight, Mw and [η] reported by Yanaki and co-workers,19 Mw is estimated to (2-3) × 106 g/mol. All chemicals used were p.a. grade. 2.3. Dynamic Oscillation Measurements. The scleroglucan sample was dissolved directly in aqueous 0.316 M NaOH (pH 13.5) for the present investigations. The sample was left stirring for 2 h at room temperature. The solution was controlled for visual impurities before adding formamide or ethyl acetate to induce the reduction in pH. The scleroglucan sample and the pH reducing agent were blended, and an aliquot was transferred to the sample stage of the rheometer (Bohlin-VOR) to determine the gelation kinetics. The rheological properties during the gelation were determined employing a serrated, parallel plate (SP30, Ø30 mm) geometry. An aliquot of 1 mL of the sample was loaded on the SP30 geometry, and the gap was adjusted to 1 mm. The sample was sealed using a low density, low viscosity silicon oil to limit potential problems associated with drying of the sample during the extended experiments. Torsion bars with a torsion stiffness of 0.235 g cm or 4.0 g cm were employed. The storage, G′, and loss, G′′, moduli at ω ) 6.28 s-1 were repeatedly determined at fixed intervals of 3 min. This interval was changed to 10 s or 10 min in some of the experiments. For simplicity data is presented with symbols for only every 5th to 30th data point. When lines are drawn between symbols, this indicates additional data points through which straight lines have been drawn without curve fitting. The strain amplitude was set to 10% of maximum in the experimental setup. This corresponds to a maximum strain less than 0.01. 2.4. Determination of the pH. The decrease in pH following addition of the pH-reducing agent to the alkaline solution of scleroglucan were determined in separate experiments using a Radiometer PHM92 lab pH meter equipped with a PHC2411 combined electrode. The pH was determined directly in solution during the in situ gelation at 20 and 50 °C. For gelation at 75 °C, aliquots sampled from the gelling solution were quenched on ice prior to determination of pH. 2.5. Stepwise Neutralization by Dialysis. A suspension of 50 mg/mL of scleroglucan in deionized water was transferred to cylinders (d ) 14 mm, l ) 15 mm) with both end surfaces covered by dialysis membrane. The cylinders containing the scleroglucan suspension were initially dialyzed against an excess of aqueous 0.316 M NaOH solution for 1 day. Subsequently, the dialysis solution was replaced with a

Aasprong et al.

Figure 2. (a) Hydroxide concentration, (b) storage modulus G′ (ω ) 6.28 s-1), and (c) loss modulus G′′ (ω ) 6.28 s-1) versus time after addition of formamide to a final concentration of 0.632 M to an aqueous 0.316 M NaOH solution of 50 mg/mL of scleroglucan at 20 (O), 50 (9), and 75 °C (4). The change in the free OH- concentration in aqueous 0.316 M NaOH (no scleroglucan added) following addition of 0.632 M formamide at 20 °C is also shown (.).

new aqueous NaOH solution once a day, each time lowering the pH by approximately 0.2. 3. Results and Discussion Figure 2 shows the free [OH-] and storage and loss moduli (G′ and G′′) at 20, 50, and 75 °C for 50 mg/mL scleroglucan in aqueous alkaline solution, respectively, versus time after addition of formamide. The scleroglucan was dissolved in 0.316 M NaOH, and the time scale depicts the time after adding formamide to a final concentration of 0.632 M. The

Scleroglucan Gelation by in Situ Neutralization

concentration of OH- was calculated from the experimentally determined pH. The data show that the [OH-] in aqueous solution starting from 0.316 M NaOH levels off in somewhat more than 10 h at T ) 20 °C following the addition of 0.632 M formamide. Achieving a stable pH reading required longer time in the presence of scleroglucan than without. Additionally, details in the time course of the pH reduction were found less reproducible when scleroglucan was included. Buffering by the scleroglucan at highly alkaline conditions may explain the somewhat lower initial pH in this case. The rheological data shows that the storage modulus increases and levels off to a plateau nearly independent of the temperature (Figure 2b). At any given time t < 1 h after initiation of the in situ pH reduction, the highest value of G′ was observed for the gel that had reached the lowest pH. This indicates that reduction of pH below a certain limit is one critical factor in forming the interchain junctions. Some insight in these aspects can be gained from the temperaturealkaline conformational phase diagram of the (1f6)-β-Dgluco-(1f3)-β-D-glucans (Figure 1).10 The process of using formamide to reduce the pH in the solution starting at 0.316 M NaOH corresponds to changing the solvent condition from that yielding the dissociated (1f6)-β-D-gluco-(1f3)-β-D-glucans to those favoring the triple-helical conformation (Figure 1). The process involves crossing the conditions where the triple-helical I prevails (at 20 °C) or crossing the same region during a shorter timeperiod (at 50 °C). In the conformation phase diagram (Figure 1), symbols are given along the pH-reduction path used experimentally for each 1/2 hour after initiating the pH reduction. This is based on the experimental data plotted in Figure 2a. At 75 °C, the process of neutralization brings the sample directly from the dispersed state to the region for the triple-helical II conformation. Significant increase in storage modulus reflecting the formation of junction zones is not expected when the [OH-] in the solution is in the region corresponding to the random coil conformation of scleroglucan. The temperature-dependent hydrolysis of formamide and thereby the rate of the pH reduction in solution is expected to be a limiting factor in induction of the gelation. At the higher temperature, a shorter time period is expected for reaching conditions favoring the triple helix. Thus, the experiments carried out at the higher temperature is expected to show a shorter initial lag time before sufficient interchain contact points necessary for macroscopic gel formation can be observed. The latter effect is similar to the existence of an induction period for the gelatin.7 An additional facet is that the kinetics of pH reduction depends on temperature, which may affect the gel modulus at a given pH in a kinetically controlled system (see below). Figure 3 shows the storage modulus versus time after starting the pH reduction of 50 mg/mL scleroglucan initially dissolved in aqueous 0.316 M NaOH. These gelation experiments were carried out for temperatures in the interval 20-90 °C. A constant molar ratio of 2 between formamide and initial NaOH concentration was used except at 90 °C where data for a ratio of 4 is shown. Increase in this molar ratio above 2 is found to have only minor influence on the

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Figure 3. Effect of temperature on gelation kinetics of scleroglucan gels formed by the in situ pH reduction of aqueous alkaline solution. Storage modulus of 50 mg/mL scleroglucan initially in 0.316 M NaOH versus time after addition of formamide to a final concentration of 0.632 M at 20 (O), 30 (b), 40 (0), 50 (9), and 75 °C (4) and to a final concentration of formamide of 1.264 M at 90 °C (2), respectively.

rheologically determined gelation kinetics (see below). At low temperatures, an initial parabolic maximum of G′ versus time was observed, whereas a monotonic increase in G′ was observed for the higher temperatures. Interpretation of the temperature effect on the gelation kinetics in these experiments is difficult because the temperature dependence on the rate of the pH reduction cannot easily be decoupled from the intrinsic temperature dependence of the molecular organization process. We note here that the two gelation experiments showing the most profound local maxima in the evolving G′ (at T ) 20 and 30 °C) are characterized by the longest duration within the triple-helical I region of the phase diagram. The reported13 increase in the viscosity in this region thus represent a possible origin for the observed maxima in G′ here. The absolute value of ∆pH/∆t increases with increasing temperature. This reduces the initial lag time of the gelation. For t > 2 h after the initiation of pH reduction, it is observed an increase in the rate of change of the storage modulus (∆G′/∆t) with increasing temperature for two regimes, temperatures from 20 to 30 °C and temperatures above 40 °C. Some similarity between the reassociation process of scleroglucan and gelatin can be expected because they both originate from triple-helical structures. Harrington and Rao20 present a schematic view of the renaturation process of gelatin. At low concentration of polymer, self-association is likely to be the dominating phenomena. This is observed for scleroglucan, e.g., with the formation of a macrocyclic topology.10,21 At high concentrations of gelatin (>10 mg/ mL), interstrand association is described as being dominating, with two processes competing, depending on the magnitude of the driving force of gelation. This driving force in thermosetting gels is the undercooling temperature, ∆T, taken as the difference between the conformational transition temperature and the gel setting temperature. With a large ∆T, the formation of a gel will be dominating, whereas a smaller ∆T results in more regeneration of linear triple helical

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Figure 4. Effect of scleroglucan concentration on gels formed upon in situ pH reduction of aqueous alkaline solution of scleroglucan at 75 °C. Initial [OH-] was 0.316 M, and formamide was added to a final concentration of 0.632 M. The storage modulus is given versus time after initiating pH reduction for the scleroglucan concentrations 10 (3), 20 (9), 30 (0), 40 ([), 50 (]), 70 (1), 100 (O), and 200 mg/ mL (b).

Figure 5. Inverse of the time to reach a fixed value of the storage modulus plotted versus scleroglucan concentration for gelation by in situ pH reduction of the aqueous alkaline solution at 75 °C. Initial [OH-] was 0.316 M and formamide was added to a final concentration of 0.632 M. Data shown for G′ ) 0.5 (O), 1 (b), 5 (4), 10 (2), 50 (0), and 100 Pa (9).

structures consisting of nearly equally long single stranded species. In this study, the driving force for gelation, and the analogue to ∆T, is the time dependent ∆pH. The observed increase in ∆G′/∆t with increasing |∆pH/∆t| could be expected as an analogue to the increase in ∆G′/∆t with increasing |∆T| observed by Nijenhuis7 in the case of gelatin. Degradation of scleroglucan at high temperature and high pH in the presence of oxygen can be expected to decrease the strength of the final network and the initial rate of gelation. Storing a 50 mg/mL scleroglucan, 0.316 M NaOH solution at 75 °C for 18 h prior to adding formamide resulted in a reduction of 3 decades of the plateau value of the storage modulus (data not shown). Prestoring at 75 °C for 40 min reduced the final modulus by approximately 20%. During the experiments at 75 °C, the [OH-] was reduced to less than 0.01 M in less than 10 min. Scleroglucan degradation can therefore not fully explain the observed difference in the plateau value of G′ at high (e.g., 75 °C) compared to lower temperatures (e.g., 20 °C). Harada and co-workers22 reported that the Curdlan gels formed from neutralizing an alkaline solution with carbon dioxide in air were much stronger than those obtained by heating at 60 °C. The reason is suggested to be the greater extent of initial strand separation in the first case. A disadvantage using formamide for in situ pH reduction is that ammonia (NH3) is one of the hydrolysis products. At alkaline conditions, the equilibrium will be shifted from NH4+ toward the less soluble NH3 (eq 3). The rate of hydrolysis and the concomitant release of NH3 are the largest at high temperature where network formation is also fast. The solubility of NH3 is also lower at high temperatures. Figure 4 shows the storage modulus versus time after initiating in situ neutralization of aqueous alkaline scleroglucan with Cp from 10 to 200 mg/mL at 75 °C. Both the

rate of change of G′ and the final storage modulus are observed to depend on the polymer concentration. Although there was hardly any increase in G′ for Cp ) 10 mg/mL during the 8 h the data were collected, an increase from less than 1 Pa to 6000 Pa was observed at a concentration of 200 mg/mL. The loss moduli (not shown) was found to change less during the gelation, yielding a reduction in the phase angle characteristic for a sol-gel transition for concentrations Cp larger or equal to 20 mg/mL. This set of data indicate a lower critical concentration needed for gelation in the range less than, but close to, 20 mg/mL. The initial rate of change of G′, a macroscopic property of the gel, was used to further characterize the gelation kinetics. Figure 5 shows kinetic parameters extracted from the initial time course of G′ at various concentrations (Figure 4). The inverse of the time needed to yield a given increase in G′ was extracted as the kinetic parameter. Linear regression of this parameter versus the polymer concentration indicates an intercept with the concentration axis at a finite Cp in the range 15-20 mg/mL. This must be the lower concentration needed for formation of 3-D network and corresponds to the lower critical concentration for gelation. Figure 6 shows the storage modulus determined 8 h after the initiation of gelation by adding formamide to the alkaline solution versus the polymer concentration. These data were extracted from the set of data obtained at T ) 75 °C (Figure 4). The time course of G′ indicates that a plateau is reached within the first 8 h. The data indicate that the exponent in a power law dependence of G′ on the concentration in the range 50-200 mg/mL is close to two and that there is a lower critical polysaccharide concentration needed for gelation. The present system shares several features with the gelation of gelatin to which the cascade model has been fitted.23,24 The cascade model was fitted to the experimental data of G′ versus Cp using the approach outlined by Clark

Scleroglucan Gelation by in Situ Neutralization

Biomacromolecules, Vol. 4, No. 4, 2003 919 Table 1. Parameters Obtained from Fitting Experimental Data to the Cascade Model for Scleroglucan Assuming M ) 1.0 × 106 g/mol and Using Varying Values of f f)5 f ) 10 f ) 15 f ) 25

a

Cp,0 [mg/mL]

K [L/mol]

9.4 5.6 4.4 3.3

15.2 14.9 14.0 12.2

5.85 × 103 9.44 × 102 3.94 × 102 1.48 × 102

This yield the following expression for the storage modulus: R Cp f - 1 G′ ) aRT (1 - ν)2(1 - β) 2K Cp,0 (f - 2)2

Figure 6. Storage modulus 8 h after initiation of in situ pH reduction plotted versus scleroglucan concentration. The data points (b) and fit (-) to the cascade model (see text for details) are shown for gels at 75 °C, and the initial [OH-] ) 0.316 M, pH reduction initiated by adding formamide to a final concentration of 0.632 M. The data point for Cp ) 10 mg/mL (O) was excluded when fitting the data to the model.

and co-workers.23,25 The storage modulus is given by 1 G′ ) {NfR(1 - ν)2(1 - β)}aRT 2

(6)

where N is the number of chains per unit volume, f is the functionality or sites per molecule available for making connection to other chains, R is the fraction of the f reacted at the actual time. Parameter ν is the extinction probability representing the probability that a given pathway extending from a given cross-link is not connected to the infinitely large (macroscopic) network. The extinction probability is given by ν ) (1 + R + Rν)f-1

(7)

and parameter β relates to the other parameters as: β)

(f - 1)Rν 1 - R + Rν

(8)

The expression in the parenthesis (eq 6) reflects the number of elastically active chains. Deviations from ideal entropy elasticity are included using the parameter a in eq 6. It has been reported that this parameter adopts values in the range from 1 to 10 for various polysaccharide gels.25 The number of moles of chains per unit volume is given by the ratio between the polymer concentration and the molar mass, N ) Cp/M. We adopt the equilibrium model detailed by Clark as a starting point for interpretation of the data. In this model, the lower critical polymer concentration needed for gelation is given by f-1 Cp,0 ) M Kf(f - 2)2

(9)

where K is the equilibrium constant for forming a junction.

(10)

The experimentally determined G′ is fitted to this expression using nonlinear regression by minimizing the sum square of residuals of the logarithm of G′ under the constraint of selected M and f.25 Independent parameters a and K are adjusted in the fitting procedure and Cp,0 is calculated using eq 9. The constrained fit to the cascade model comply with the experimentally determined trend of G′ from Cp ) 20-200 mg/mL (Figure 6). The fits of the cascade model (Figure 6) were carried out using a molar mass corresponding to onethird of the Mw of the triple-helical state, and using various f. Physically reasonable values of f are not straightforward to estimate. The cooperative length of the strand-separation transition determined at high temperature is reported to be in the order of 100 repeating units.8 This indicate an f of about 15 for M ) 1 × 106 g/mol. An upper value of f for the same molar mass was estimated to f ) 25 from the transition from triple-helices above about M ) 40 × 103 g/mol, dissociating to single strands at shorter chain lengths.26 Data for the alkaline induced strand separation transition are more difficult to use because of the large difference in enthalpy associated with conformational transition and depolymerization encountered at high pH.10 The results show that the experimental data can be accounted for using a cascade model with f in the range 5-25 (Figure 6). The various f yield estimates of a lower critical concentration Cp,0 in the range 12-15 mg/mL (Table 1), which is in agreement with the range estimated based on the initial gelation kinetics (Figure 5). Note that the G′ obtained at Cp ) 10 mg/mL is above the expected values of the fit for most selections of f. The estimated values of the front-factor a, 3-9 (Table 1), is in the range reported for other polysaccharides.25 Reduction of the molar mass to 0.5 × 106 g/mol in the model calculations yielded equally good fits (graphical data similar to those in Figure 6). The obtained Cp,0 are within the same range as obtained for a molar mass of 1.0 × 106 g/mol. Preference for either of these molar masses, in the interpretation of the data, in terms of the cascade model cannot be established. These molecular weights are both within the central part of a molar mass distribution with Mw ) 1.0 × 106 g/mol. The reported macrocyclic species being formed when the same conformational boundaries are crossed at lower scleroglucan concentrations10,21 indicate that an irreversible cascade description may be more appropriate than the reversible.23,25

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Figure 7. Frequency dependence of storage (open symbols) and loss (filled symbols) moduli during gelation of 50 mg/mL scleroglucan by the in situ pH reduction of the aqueous alkaline solution at 20 °C. The initial concentration of [OH-] was 0.316 M. The frequency sweeps are shown for the following time intervals after initiating gelation by adding formamide to a final concentration of 0.632 M: 42 min (3,1), 3.5 h (4,2), 6.5 h (O,b), and 65 h (0,9).

The storage modulus versus concentration is then described in terms of a kinetic model where K represents the ratio between associations leading to wastage of material in building an elastically active network, e.g., cyclization, and those yielding effective cross-linking. Additional experiments where the pH of a 50 mg/mL scleroglucan solution was reduced stepwise and slowly from pH 13.5 was therefore carried out using dialysis. Mobile air bubbles in the stepwise pH-reduced solutions down to pH 12 indicated a liquid nature. Moreover, further stepwise dialysis decreasing pH to 11.1 indicated a thickening of the solution but no gel being formed. A large influence of degradation was ruled out because increasing the pH to 13.5 and quickly reducing the pH produced gels. This finding indicates that the rate of lowering the pH is important for the final gel strength. Recently, a model including bending of stiff, reconstituted gelatin junction zones was launched as an alternative to the previous cascade model of gelatin.27 This represents an interesting idea that also can be explored as an alternative mechanism for storing the deformation free energy during mechanical testing of the present scleroglucan gels, in particular when additional molecular detail of the reconstituted triplex fraction in the gel network is elucidated. The observed changes in the mechanical spectra during the gelation are shown in Figure 7. The frequency dependence of G′ and G′′ after 42 min is characteristic for that of a solution. After 3.5 h the storage modulus has increased to a level above that of the loss modulus, and displays a power law in the frequency dependence similar to that of the loss modulus. Later in the gelation process, the storage modulus is found to increase in absolute value and to become less dependent on frequency approaching a plateau behavior. In contrast, G′′ is relatively less changed. This series of mechanical spectra indicate that the in situ reduction of pH

Aasprong et al.

Figure 8. Effect of concentration of the pH-reducing agent, formamide, on gelation of a 50 mg/mL aqueous alkaline solution of scleroglucan at 75 °C. The scleroglucan was dissolved in 0.316 M NaOH and gelation was initiated by adding formamide to give the ratios [Formamide]/[NaOH] initial: 0 (3), 0.5 (O), 1 (0), 2 (4), and 5 (9).

induces a sol-gel transition going through the stages where critical gel behavior can be identified according to the Winter-Chambon criteria. Figure 8 shows the increase in the storage modulus versus time after initiation of the neutralization using various molar ratios of formamide to sodium hydroxide. The data show that the ratio of formamide to hydroxide ) 1:2 only yield a storage modulus of 0.5 Pa within the first 12 h, whereas in stoichiometric ratios, a plateau value of G′ is reached within the same time period. Increase in the formamide concentration above the stoichiometric ratio does not increase the equilibrium modulus but yields a small increase in the initial rate of G′. These series of experiments indicate the need for a stoichiometric amount of neutralizing agent as compared to the initial [OH-] to reduce the pH sufficiently for gelation to be achieved. It is also evident that excessive concentration of neutralization agent is not necessary. The gelation kinetics can be controlled by selection of the pH-reducing agent. Figure 9 shows the storage modulus versus time for 50 mg/mL scleroglucan initially dissolved in 0.316 M NaOH where 0.632 M of either formamide or ethyl acetate were employed to reduce the pH in the solution. The data both at 50 and 75 °C show a larger initial rate of change of G′ when ethyl acetate was used as the in situ pHreducing agent then for formamide. The increase in the storage modulus within the first 2 h (∆G′/∆t)0-2h at 50 °C increased from 80 to 125 Pa h-1 when substituting formamide with ethyl acetate, both at 0.632 M. At 75 °C, the same values were 200 and 350 Pa h-1. Although a limited set of compounds has been applied in this series of experiments (Figure 9), it does provide evidence that the kinetic of gelation can be controlled by selection of the hydrolyzing agent. We have carried out a series of experiments for gelation of scleroglucan utilizing the triple-helical motif of individual β(1f3)-D-glucan chains by dissolution of the samples at conditions favoring triple-helical dissociation at high pH and

Scleroglucan Gelation by in Situ Neutralization

Figure 9. Comparison of the effect of formamide and ethyl acetate as the pH-reducing agent on the gelation kinetics of a 50 mg/mL aqueous scleroglucan solution initially at [OH-] ) 0.316 M. Storage modulus versus time after initiating pH reduction by adding the pHreducing agent to a final concentration of 0.632 M is given at 50 (filled symbols) and 75 °C (open symbols) for ethyl acetate (O,b) and formamide (0,9).

adding a chemical compound that hydrolyses yielding a reduction of pH with concomitant association of the chains. Selection of pH-reducing agent, concentration, and temperature controls the gelation kinetics and the final gel properties. Acknowledgment. This work was supported by VISTA (grant V6312), a research cooperation between the Norwegian Academy of Science and Letters, and Statoil. We gratefully acknowledge Sanofi Bioindustries, France for providing the scleroglucan sample. References and Notes (1) Crescenzi, V.; Imbriaco, D.; Velasquez, C. L.; Dentini, M.; Ciferri, A. Macromol. Chem. Phys. 1995, 196, 2873-2880.

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