GELATION OF KGM IN THE PRESENCE OF ALKALI

3.4 GELATION OF KGM IN THE PRESENCE OF ALKALI

3.4.1 G ELATION K INETICS OF KGM WITH D IFFERENT M OLECULAR W EIGHTS

The time dependence of G' and G'' or 2% aqueous dispersions of KGM with molecular weights from 2.56 to 5.96 × 10 5 in the presence of 0.02 mol/l sodium carbonate at 60˚C is shown in Figure 3.7. 19 Time t = 0 was defined as the time when the alkali was added. The observed curve for G' as a function of time was fitted well by an equation for the reaction of the first order. The gelation time t 0 , at which G'

´/Pa G 10 2

Log 1

2 G˝/Pa

10 Log 1

t/min

FIGURE 3.7 Time dependence of G' and G'' for 2% aqueous KGM dispersions with different molecular weights. ▫, LM1; 〫, LM2; 䉭, LM3; × , LM4. Measurement temperature: 60˚C. Symbols represent the experimental values and the solid lines represent the calculated curves. (From Yoshimura, M. and Nishinari, K., Food Hydrocoll., 13, 227, 1999. With permission. )

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began to deviate from the baseline, became shorter and the saturated G' increased with increasing molecular weight. The time dependence of G' and G'' for aqueous

dispersions of KGM with the molecular weight of 2.56 × 10 5 and different concen- trations from 1.0 to 3.0% in the presence of 0.02 mol/l sodium carbonate at 65˚C

was also fitted well by an equation for the reaction of the first order (data not shown). The gelation time t 0 became shorter and the saturated G' increased with increasing

concentration of KGM. In the time dependence of G' and G'' for 2 and 3% aqueous dispersions of KGM with the molecular weight of 4.38 × 10 5 at temperatures from 40 to 90˚C in the presence of 0.02 mol/l sodium carbonate, the maximum of G' was observed at temperatures above 75˚C. 19,20 To examine whether the maximum of G' is a real phenomenon or an artifact caused by the slippage, the penetration force was

measured using a spherical Teflon plunger of radius 5 mm. 20 This method has the advantage that it is free from slippage. The normalized penetration force observed

at different temperatures from 50 to 90˚C did not show any maximum as a function of time, indicating that the maximum observed in G' by the oscillatory shear mode

mentioned above was induced by the slippage. The apparent maximum in G' in the study of gelation is a notorious problem and has sometimes been reported erroneously. 20

The time dependence of G' and G'' for aqueous dispersions of KGM with the molecular weight of 1.17 × 10 6 , which was not degraded by enzyme, with different concentrations from 0.5 to 2.0% in the presence of 0.02 mol/l sodium carbonate at 80˚C did not show any maximum. The gelation time t 0 became shorter and the saturated G' increased with increasing concentration, as in enzymatically treated samples. The slippage may be due to disentanglement of the adsorbed chains of KGM during gelation. When the surface of the fixture is predominantly occupied by the free water molecules, catastrophic slippage takes place. 20

A molecular level description of the time course of the gelation of KGM was presented by Williams et al., 21 and the role of alkali addition was considered in detail. Nuclear magnetic resonance (NMR) relaxometry was utilized as a comple- mentary methodology to mechanical spectroscopy to probe events occurring as a prelude to network formation, and high-resolution NMR was used to follow the deacetylation process. It was shown that the addition of alkali plays an important solubilizing role as well as facilitating the deacetylation of the chain. Deacetylation is important both in reducing the inherent aqueous solubility of the polymer and in progressively negating the alkali-induced polyelectrolytic nature of the polysaccha- ride chain via reaction-induced pH changes. Figure 3.8a shows spin–spin relaxation

time T 2 obtained from 1% KGM samples after the addition of alkali, measured during a series of temperature ramps (at 1˚C min –1 ) from 20˚C to 50, 60, 70, and

80˚C and subsequent holding periods. The initial linear-like response merely reflects the temperature dependence of the chain mobility, although it should be remembered

that at this concentration there is an initial solubilizing effect of alkali. After this initial period (some 25 to 30 min of ramping at 1˚C min –1 ), it is clear that the

observed T 2 begins to drop. The simplest interpretation here is that the chain mobility now begins to decrease owing to the aggregation of material that has undergone

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alkali-induced deacetylation. It is clear from the rheological data shown in Figure 3.8b that with a continued temperature increase, the modulus increases with the rate dependent on the holding temperature. There also seems to be some evidence of a delay period at the lower temperature (50˚C) after the final temperature is reached before the elastic modulus is detected to rise.

It is proposed that observed induction periods following alkali addition (during which the elastic modulus does not rise) are not simply deacetylation delays but are related to the aggregation kinetics of the deacetylated material, although the two processes may appear indistinguishable. A summary of the proposed mechanism is given in Figure 3.9.

3 15 50 T/°C 50

2 G´/Pa 10

T 2 /s 0 0

0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90 t/min

t/min FIGURE 3.8 (a) T 2 values obtained from 1% KGM samples after the addition of alkali,

measured during a series of temperature ramps (at 1˚C min –1 ) from 20˚C to 50, 60, 70, and 80˚C and subsequent holding periods. (b) Corresponding elastic modulus (measured at 1 Hz and 0.5% strain) for a 1% KGM solution under the same thermal regime as the NMR experiments (a) with identical alkali addition. In both cases, the data are given in the same symbols as the corresponding temperature ramp (also shown) under which they were obtained. (From Williams, M.A. et al., Biomacromolecules, 1, 440, 2000. With permission.)

Entangled chains +OH − Increase solvation

Deacetylation Decrease inherent chain solubility

Decrease polyelectrolytic solubility Aggregation

Produce nuclei Percolation

Gel formation

FIGURE 3.9 Schematic diagram of the proposed gelation mechanism for KGM. (From Williams, M.A. et al., Biomacromolecules, 1, 440, 2000.)

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To understand the role of acetyl groups in the gelation of KGM solutions, several fractions of KGM with different degrees of acetylation were prepared. 17 Five frac- tions with different acetylation levels were obtained. The degree of acetylation (DA) (the weight percent of acetyl-substituted residues in KGM backbone) ranged from

1.6 (not treated) to 5.3%. The molecular weight determined by light scattering in cadoxen was decreased to about the half value by the deacetylation. 17 However, the various acetylation reaction conditions, such as different acetylation temperature and the amount of catalyst, did not lead to a remarkable difference in molecular weight. Therefore, a slight difference in molecular weight between the acetylated samples was not taken into account, and the attention was paid mainly to the influence of DA. The mech-

anism of the main chain scission was explored later by Gao and Nishinari 23 to get better or ideal samples with different DAs without changing the molecular weight. Figure 3.10a shows the time dependence of G' of 2.0 wt% aqueous dispersions of KGM with different DAs in the gelation process at 45˚C in the presence of Na 2 CO 3 . The concentration of Na 2 CO 3 was fixed as 0.2 wt%. The value of G' at t = 0 for a native fraction Rs was far larger than that for acetylated KGM samples. However, G' of Rs was finally overtaken by that of acetylated KGM samples with time elapsing. The gelation time, t gel , defined as the time of the crossover of G' and G'', as shown in Figure 3.10b, became longer with increasing DA. Although the gelation time determined in this way slightly changed with the frequency, this method

was adopted for the simplicity. 22 It is expected that the deacetylation reaction and further aggregation process for KGM samples with higher DAs need a longer time than those for KGM with lower DAs. The G' of all the samples increased rapidly in the beginning of gelation and finally attained the plateau values. It took a longer time for KGM with higher DAs to reach the saturated value of G'. The G' of the native KGM, Rs, attained maximum values in ca. 245 min (ca. 4 h), while the G' of a KGM fraction with the highest DA, Ac32, still continued to increase even after 2300 min (ca. 38 h). The reason why Ac32 showed a different behavior is interpreted

Ac20 Ac21

Ac26 Ac27 Ac31

o g G´/Pa 1.4 2.4 t gel L 60

3.4 /min

0.4 o 0.4 L g G´/Pa –0.6

t/min 100 150

t/min DA/% (a)

(b)

FIGURE 3.10 (a) Time dependence of G ' of 2.0 wt% aqueous dispersions of KGM with different DAs in the gelation process at 45˚C in the presence of Na 2 CO 3 . The concentration of Na 2 CO 3 was fixed as 0.2 wt%. (b) Gelation time t gel of the KGM aqueous dispersions against DA. (From Huang, L. et al., Biomacromolecules, 3, 1296, 2002. With permission.)

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as follows: the DA of Ac32 is the highest, and the largest amount of alkali is required. The present concentration of Na 2 CO 3 solution was insufficient for 2 wt% Ac32 dispersion to form a gel in a short time. It was difficult to observe the exact plateau values of G' for some acetylated KGM samples in this experimental condition due to the long measuring time.

After a certain amount of time (data not shown), both storage and loss moduli, G' and G'', of Rs aqueous dispersions, in the presence of Na 2 CO 3 at a fixed concen- tration ratio of Na 2 CO 3 to the degree of acetylation of KGM (0.1), were found to increase monotonically and attained plateau values, the saturated storage modulus (G' sat ) and loss modulus (G'' sat ) respectively, as was observed previously. t gel increased sharply with decreasing concentration, and G' sat increased with increasing concen- tration. Since molecular chains are close to each other at higher concentrations, the probability of the formation of a junction zone is higher than that at lower concen- trations. Gelation would begin even before the complete loss of acetyl groups at higher concentrations; therefore, t gel became shorter with increasing concentration of KGM, as expected. 17

If the alkaline concentration (C Al ) to DA was fixed to a constant (0.1), a similar time course of G' for all the samples, except the fraction with the highest DA, would

be observed as shown in Figure 3.11. It indicates that C Al /DA plays a crucial role in the gelation process. The gelation rate is determined mainly by the ratio of alkaline concentration to the degree of acetylation. However, the fact that Ac32 shows an exceptional behavior should be explored in the near future.

Recently, KGM samples with different degrees of acetylation were prepared without changing the molecular weight. 23 The acetylation of KGM was carried out using acetic anhydride in the presence of pyridine as the catalyst. By changing the reaction temperature or the amount of pyridine, a series of acetylated samples with

3 o g G´/Pa

DA = 1.6%, C of Na 2 CO 3 = 0.16% L

Rs.

Ac20. DA = 2.2%, C of Na 2 CO 3 = 0.22% 2 Ac21. DA = 2.6%, C of Na 2 CO 3 = 0.26% Ac26. DA = 2.9%, C of Na 2 CO 3 = 0.29% Ac27. DA = 4.0%, C of Na 2 CO 3 = 0.40% Ac32. DA = 5.3%, C of Na 2 CO 3 = 0.53%

t/min

FIGURE 3.11 Time dependence of G' of 2.0 wt% KGM aqueous dispersions in the presence of Na 2 CO 3 at 60˚C. The ratio of alkaline concentration to the degree of acetylation was fixed to a constant (0.1). (From Huang, L. et al., Biomacromolecules, 3, 1296, 2002. With permission.)

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a DA range from 1.38 to 10.1 wt% was obtained. Table 3.1 lists the reaction conditions and some parameters of the acetylated KGM samples. The intrinsic viscosity of native KGM (Rs) decreased by about 9% from 557 cm 3 g –1 of Rs to 500 ± 20 cm 3 g –1 of each acetylated sample. Comparably, the intrinsic viscosity of native KGM decreased by 28% after acetylation by using zinc chloride as the catalyst in a previous work. 17 A slight decrease in the viscosity-average molecular weight from 12.0 × 10 5 of native KGM to 9.70 to 11.0 × 10 5 of acetylated KGM products was observed. This indicates that pyridine is a milder catalyst than zinc chloride in the acetylation of KGM.

The effect of the degree of acetylation (DA) on the gelation behaviors upon addition of sodium carbonate to native and acetylated KGM samples was studied by dynamic viscoelastic measurements. 23 At a fixed alkaline concentration (C Na ), both the critical gelation times (t cr ) and the plateau values of storage moduli (G' sat ) of the KGM gels increased with increasing DA (shown in Figure 3.12). However, at a fixed ratio of alkaline concentrations to values of DA (C Na /DA), similar t cr and G' sat values independent of DA were observed (shown in Figure 3.12). On the whole, increasing KGM concentration or temperature shortened the gelation time and enhanced the elastic modulus for KGM gel.

In conclusion, it was suggested that the deacetylation leads to the aggregation of stiffened molecular chains. In the presence of excessive alkali, the gelation proceeds too fast, resulting in a gel with a smaller elastic modulus. The final elastic modulus of gels depends strongly on the gelation rate. The KGM gel is thermoir- reversible and the rearrangement of network chains does not seem to occur as in cold-set gels, such as gellan gels and κ-carrageenan gels. It was reported recently that the helix–coil transition, which occurs in self-supporting gels of gellan induced

by temperature change, 24 also occurs in κ-carrageenan gels by immersion in salt solutions. 25 In the gelation of KGM, the gelation rate was shown to be governed by the concentration and molecular weight of the polymer, temperature, and DA and

TABLE 3.1 Effect of the Amount of Pyridine and Temperature on the Extent of Acetylation and the Results of Viscosity Measurements of the Products

Sample Rs

Ac5 Ac6 Ac-D

Pyridine (ml) — 0.5 1 1.5 2 2.5 2.5 10 Temperature (˚C)

— 40 40 40 50 50 80 40 Time (h)

— 2 2 2 2 2 2 3 DA (%)

1.38 4.13 4.47 4.82 5.85 7.40 7.57 10.15 DS

0.05 0.16 0.18 0.19 0.23 0.30 0.31 0.42 [ η] a (cm 3 g –1 )

12.0 10.1 10.9 9.88 10.3 11.0 9.91 9.70 a The viscosity measurements of KGM cadoxen solutions were carried out at 25 ± 0.02˚C by using an Ubbelohde-type viscometer. The viscosity-average molecular weights (M v ) of the KGM samples were

calculated according to [ η] = 3.55 × 10 –2 M 0.69 . 8

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FIGURE 3.12 Plots of t cr of the KGM samples as a function of DA in the case of C Na /DA = 0.2 (solid square) and C Na /DA = 0.1 (solid circle) and in the case of the concentration of sodium carbonate (C Na ) as 0.8 wt% (hollow circle). (From Gao, S. and Nishinari, K., Biom- acromolecules , 5, 175, 2004.)

alkaline concentration, and it was also shown that slower gelation leads to the stronger gels. Although molecular forces responsible for gel formation in both cold- set gels and KGM gels are believed to be hydrogen bonds, tighter stacking occurs in KGM gels than in other thermoreversible gels, such as gellan, carrageenan, agarose, etc. Stacking might be tighter in solutions of polymers with longer persistent length. Unfortunately, the persistent length of KGM is not known because of its poor solubility in water. The atomic force microscopy (AFM) observation will be useful to gain insight on this point.