Biomacromolecules 2003, 4, 1623-1629
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Characterization of Association and Gelation of Pectin in Methanol-Water Mixtures Ingunn Tho,† Anna-Lena Kjøniksen,‡ Bo Nystro¨m,*,‡ and Jaan Roots‡ Department of Pharmaceutics, School of Pharmacy, University of Oslo, P.O. Box 1068, Blindern, N-0316 Oslo, Norway, and Department of Chemistry, University of Oslo, P.O. Box 1033, Blindern, N-0315 Oslo, Norway Received March 4, 2003
Turbidity, swelling, and rheological features of semidilute systems of pectin in methanol-water media of different composition have been investigated. By increasing the percentage of methanol in the mixture, the thermodynamic properties of the pectin/methanol/water system become poorer, as shown by increasing turbidity and decreasing swelling. Effects of oscillatory and steady shear flows on intermolecular associations and gelation of pectin in methanol/water mixtures are reported. The effects of methanol concentration on the growth and structure of shear-induced gels, stabilized through hydrogen bonds, are analyzed. Steady shear measurements on these systems reveal shear thickening at low shear rates and disruption of intermolecular associative junctions at high shear rates. Introduction Pectin, extracted from cell walls in most plants, is a biocompatible anionic polysaccharide that is widely used as thickener, gelling agent, stabilizer, and emulsifier in many food products. Pectins (see Figure 1) consist primarily of (1 f 4)-linked R-D-galacturonyl units occasionally interrupted by (1 f 2)-linked R-L-rhamnopyranosyl residues.1 Depending on the degree of methoxylation (DM), pectins are usually classified into high-methoxyl (HM) pectin (methoxyl content > 50%) and low-methoxyl (LM) pectin (methoxyl content < 50%). It has been shown1-3 that the degree of methoxylation governs the type of interactions operating in semidilute and gelling aqueous solutions of these systems. In the case of HM pectin (higher hydrophobicity), intermolecular associations are governed by both hydrogen bonds and hydrophobic interactions, whereas for LM pectin, hydrogenbonded intermolecular complexes are expected to play a prominent role. Both types of pectin form gels in the semidilute regime, but the gels are usually formed under different conditions. Gelation of HM pectin is usually observed at acid pH (lower than 3.5) in the presence of a large amount of sugars or similar cosolutes,4-7 which are known to reduce water activity.5,8 In the case of LM pectin, gels are formed4,6 in the presence of Ca2+, which acts as a bridge between pairs of carboxyl groups of different pectin chains, over a wide range of pH. Gelation of the latter type of pectin has also been detected9 upon cooling under acidic conditions in the absence of Ca2+. In a previous study,10 thermoreversible gelation of aqueous acid mixtures of LM pectin and chitosan was reported. Quite recently,11 a special feature of aqueous solutions (the polymer was dissolved in water, pH ) 2.8, without any adjustment of pH) of a LM * To whom correspondence should be addressed. † Department of Pharmaceutics. ‡ Department of Chemistry.
Figure 1. Schematic illustration of the principal structure of pectin.
pectin fraction (the same fraction is used in the present work) was revealed, namely exposure of the solution to oscillatory or steady shear flows may induce gelation. It was argued that alignment and elongation of polymer chains promoted the formation of the gel network, which is stabilized through hydrogen bonds. It was shown that the ability to form a shearinduced gel was lost in the presence of the hydrogen-bondbreaking agent urea. This finding supports the conjecture that hydrogen bonds are important for the shear-induced gelation in this pectin-water system. Pectin has attracted attention as an excipient for drug delivery systems. The gelling of pectin can be taken advantage of in the creation of diffusion barriers intended for delayed/retarded drug release.12-14 By varying the properties of microparticles prepared by cross-linking pectin with Ca2+, it was possible to control the drug release.15,16 Pectin in combination with other polymers is currently being investigated as a potential film forming excipient to protect drug substances in a core or to regulate drug release from the core.17-19 Recently, a technique called extrusion/ spheronization20-22 has been utilized in the preparation of pectin pellets. To act as a successful excipient in the extrusion/spheronization process, the swelling of the polymer must be under control. Different approaches to tune the swelling properties of pectin during the extrusion/spheronization have been
10.1021/bm0300204 CCC: $25.00 © 2003 American Chemical Society Published on Web 10/15/2003
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investigated.20-22 The most successful method is to change the solvent power of the granulation liquid used together with pectin during extrusion. This can be accomplished by employing methanol-water mixtures as granulation liquid. By increasing the amount of methanol in the mixture, the solvent power deteriorates and the swelling of pectin is eventually arrested. To gain further insight into the factors that govern the swelling of pectin and how the physical properties of the system are affected, we have undertaken an extensive study, employing several experimental techniques, of the physicochemical behavior of pectin in methanol-water media of different composition. One objective of this investigation is to provide basic knowledge of the physical features to better understand the underlying mechanisms operating during extrusion/spheronization of such systems. The experimental methods include turbidimetric measurements, swelling experiments, oscillatory shear, and steady shear measurements. Experimental Section Materials and Solution Preparation. A LM pectin sample, Pectin Classic CU701 (lot. no. 0903185), was obtained from Herbstreith & Fox GmbH, Germany. According to the specifications from the manufacturer, this sample has a DM of 35% and the galacturonic acid content is 88%. The pectin was produced from citrus peels. The molecular weight was determined by capillary viscometry on dilute solutions of pectin dissolved in 1 wt % sodium hexametaphosphate (NaPO3)6 at 20 °C with the following MarkHouwink equation [η] ) 9.55 × 10-2 M0.73 mL/g. The molecular weight was estimated from intrinsic viscosity data to be 5 × 104. The molecular weight distribution of the sample is not known, but it is probably broad. To remove impurities, samples of the raw materials were centrifuged for 12 h at 3800 rpm and dialyzed against deionized water for 7 days and freeze-dried prior to use. As a dialyzing membrane, regenerated cellulose with a molecular weight cutoff of 8000 (Spectrum Medical Industries) was used. The other chemicals utilized in this study, glutaraldehyde (Fluka) and cyclohexyl isocyanide (Fluka), were used without further purification. The freeze-dried polymer was dissolved in water or in methanol/water mixtures, and solutions with a total polymer concentration of 1 wt % were prepared. The pH of these solutions is around 3, and prior to measurement, the solutions were allowed to equilibrate for 12 h. All the experiments were carried out on solutions at 25 °C, except the swelling measurements, which were carried out at ambient temperature. The swelling experiments were conducted on crosslinked pectin networks, which were prepared following the Passerini three-component condensation23 as described by de Nooy and co-workers.24,25 In the Passerini condensation, a carboxylic acid and a carbonyl are condensed with an isocyanide resulting in formation of an R-(acyloxy)-amide. In the preparation of the cross-linked pectin networks for the swelling experiments, pectin solutions of 3 wt % were heated at 60 °C to obtain low-viscous solutions (pH was approximately 3). 10.8 µL (0.125 mmol) glutaraldehyde
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followed by ca. 40% excess of cyclohexyl isocyanide (21.5 µL; 0.175 mmol) was added to 15 g of the pectin solution. This gives, based on the galacturonic acid content in the used pectin (DM 35), a theoretical cross-link density of ca. 6%. The reaction was carried out at 60 °C to keep the viscosity of the solution low enough to allow good mixing of the components. As the viscosity of the solution increased, normally after 2-3 min, the sample was poured into a Petri dish. The film that was formed was allowed to cure overnight before it was washed in deionized water and dried in a vacuum oven at 40 °C for 7 days. Turbidity Measurements. Turbidity of pectin (1 wt %) in methanol/water mixtures of different composition was measured with a Medispec II (Medinor Produkter, Norway) spectrophotometer at a wavelength of 600 nm and the transmittance was recorded. The results are reported in terms of the reduced transmittance T/T0, where T is the transmittance at a considered methanol/water ratio and T0 is the value in the pectin/water solution. Swelling Experiments. The equilibrium degree of swelling of pectin in methanol/water mixtures of different composition was determined at room temperature. It was confirmed that a period of 4 h was more than adequate to ensure attainment of swelling equilibrium. The film samples were approximately 20 × 20 mm and the weights of the samples were approximately 20 mg. The films were accurately weighed and placed in Petri-dishes containing 30 g of the methanol-water media, ranging from 0 to 25 wt % methanol, under consideration. At regular intervals the samples were withdrawn from the test medium, blotted with a tissue to remove excess liquid, and weighed and replaced in the medium. The procedure was repeated until constant weight was obtained. The degree of swelling Gs is given by the following expression Gs )
Wt - W0 W0
(1)
where Wt is the weight of the swollen film and W0 is the initial weight of the film. The results are reported in terms of the normalized degree of swelling, which is determined by dividing the values of Gs in methanol-water media with Gs obtained in pure water. The results at each methanolwater composition are an average of 4-6 tested film samples. Rheological Experiments. Oscillatory shear and steady shear measurements were conducted at 25.00 ( 0.01 °C in a Paar-Physica MCR 300 rheometer using a cone-and-plate geometry, with a cone angle of 1° and a diameter of 75 mm. To ensure good reproducibility of the measurements and to avoid shear effects on the solutions, great care was exercised when the sample was introduced into the rheometer. Therefore, all of the solutions were transferred to the plate of the measuring cell by very slow pouring, and were allowed to equilibrate for about 10 min before commence of the experiments. To prevent dehydration from the solution, the free surface of the sample was always covered with a thin layer of low-viscosity silicone oil (the viscoelastic response of the sample is virtually not affected by this layer). The measuring device is equipped with a temperature unit (Peltier
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Figure 3. Time evolution of the dynamic moduli during sequential repeated measurements of G′ and G′′ at an angular frequency of 5 rad/s and at a fixed strain of 1% for pectin (1 wt %) in mixtures of methanol and water at different composition.
Figure 2. (a) Effect of methanol on the reduced transmittance of the pectin (1 wt %)/methanol/water system. (b) Effect of methanol on the normalized degree of swelling of pectin/methanol/water films (see text for more details).
plate) that gives a very good temperature control over an extended time. Results and Discussion Turbidity and Swelling. Figure 2a shows the effect of methanol concentration on the reduced transmittance for the pectin/methanol/water system. It is evident that the reduced transmittance is almost constant up to ca. 5 wt % of methanol, whereas at higher methanol concentrations the turbidity increases monotonically. The enhanced turbidity reflects that the thermodynamic conditions become poorer as the concentration of methanol in the mixture increases. Methanol is a poor solvent for pectin and this results ultimately, at a sufficient high concentration of methanol, in phase separation with a solvent rich and polymer rich phase. The turbidity of the samples is not observed to change with time. Figure 2b illustrates the effect of methanol on the normalized degree of swelling for a chemically cross-linked pectin film (cf. the Experimental Section). The error bars indicate the experimental accuracy, and it is shown that the experimental errors are less pronounced as the swelling is reduced. Above ca. 5 wt % of methanol, the swelling of the film falls off monotonically as the methanol percentage rises and the solvent power becomes poorer. This is a typical hallmark for a film or gel under poor thermodynamic conditions with weak excluded volume effects. In this case, the up-take of solvent to the film is suppressed. These results are qualitatively consistent with the above-reported turbidity measurements. We may note that these experiments have
been carried out at a fixed cross-linking density (ca. 6%), but the cross-linking density should affect the degree of swelling. A higher cross-linking density of the network is expected to reduce the swelling ability of the film. Both the turbidity and swelling results reveal that the thermodynamic conditions of the pectin/methanol/water system become appreciably poorer at methanol concentrations above 5 wt %. Oscillatory Shear Measurements. Time evolution of the storage (G′) and loss (G′′) modulus at a low angular frequency (ω) (5 rad/s) is depicted in Figure 3 for pectin (1 wt %) in methanol/water mixtures of different composition. The measurements were periodically repeated (every 16 min) at a small strain of 1% over an extended period of time. A conspicuous feature is the marked increase of the dynamic moduli with time. At all of the studied methanol/water compositions, the elastic response is dominating (G′ > G′′ and the moduli are nearly independent of frequency in the considered experimental window (0.5-5 rad/s)), even at the start of the experiment, and this effect is strengthened as time passes and a gel is formed. The general trend is that at moderate percentage (0-6 wt %) of methanol in the mixture, the values of the dynamic moduli increase as the concentration of methanol increases. These results suggest that moderate amounts of methanol promote the evolution and strength of the gel network. An inspection of the storage modulus data at the highest concentration (10 wt %) of methanol reveals that the values drop below the corresponding ones at 6 wt % methanol. This trend probably reflects the close proximity to phase separation at 10 wt % methanol with strong contraction of the polymer chains and thereby a weakening of the elastic response of the network. In a recent rheological study11 on semidilute pectin (DM 35)-water solutions, shear-induced gelation was reported for the first time. The gelation effect was attributed to alignment and stretching of the polymer chains, resulting in more sites that become available in the formation of intermolecular hydrogen-bonded complexes. In this process, the polymer chains hydrogen-bond to each other to form an interconnected three-dimensional gel network. It has been argued26
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Figure 4. Time evolution of the complex viscosity during sequential repeated measurements of G′ and G′′ at an angular frequency of 5 rad/s and at a fixed strain of 1% for pectin (1 wt %) in mixtures of methanol and water at different composition. The inset plot illustrates the effect of methanol on the complex viscosity at the start of the experiment and 10 h later (see text for more details).
that this may lead to structures as if the chains have been “zipped” together to form staggered ladder-like structures. Although the pectin sample considered in this work should contain fairly few hydrophobic moieties (methylated carboxyl groups), it is possible that large shear-induced deformations of the network organization make more hydrophobic “stickers” accessible to intermolecular associations and thereby contribute to the build-up of the gel network. This effect may even be more effective at low concentrations of methanol due to poorer thermodynamic conditions. In light of this, it is possible that small amounts of the additive methanol facilitate the evolution of intermolecular hydrophobic associations, but it is also possible that methanol has a favorable influence on the formation of hydrogen-bonded complexes. Figure 4 shows the growth of the complex viscosity η* |η*(ω)| )
xG′2 + G′′2 ω
(2)
(at an angular frequency of 5 rad/s) with time for the pectin (1 wt %)/methanol/water system at different methanol/water compositions. A salient feature is the pronounced progressive increase of η* as the methanol concentration increases up to 6 wt %; above this concentration, the increase of η* is retarded. The inset plot illustrates the values of η* at the start and after 10 h for the pectin/methanol/water system as a function of the methanol concentration. The behaviors of η* at different times are similar and the enhancement of the viscoelasticity is most accentuated up to 6 wt % methanol. The shear-induced gel point of a pectin/methanol/water system can be determined by the observation of a frequency independent value27 of tan δ ()G′′/G′) obtained from a multifrequency plot of tan δ versus elapsed time (see Figure 5). An alternative method28 to establish the gel point is to plot against time the “apparent” viscoelastic exponents n′ and n′′ (G′∼ωn′; G′′∼ ωn′′) calculated from the frequency dependence of G′ and G′′ at each time of measurement and observing a crossover where n′ ) n′′ ) n (see insets of Figure
Figure 5. Illustration of methods for the determination of oscillatory shear-induced gel point for the pectin/methanol/water system at two different methanol/water compositions. Viscoelastic loss tangent as a function of time for the angular frequencies indicated. Tanδ becomes independent of frequency at the gel point. The inset plots to the left show power law behaviors of the dynamic moduli at the times of gelation. At the gel point, G′ and G′′ have the same frequency dependency (G′ ∼ G′′ ∼ ωn). The inset plots to the right show changes of the apparent exponent n′ for the storage and n′′ (G′ ∼ ωn′, G′′ ∼ωn′′) for the loss moduli during the course of gelation. n′ ) n′′ ) n at the gel point.
5). Both methods yield the same gelation times for the considered pectin/methanol/water systems that have been subjected to periodical oscillatory shear perturbations over extended time. A power law between dynamic moduli and frequency given in the following way29 can characterize the rheological behavior of an incipient gel G′ ) G′′/tanδ ) SωnΓ(1 - n) cos δ
(3)
where Γ(n) is the Legendre gamma function, n is the relaxation exponent, and S is the gel strength parameter,30 which depends on the cross-linking density and the molecular chain flexibility and is considered to be a material parameter. The phase angle (δ) between stress and strain is independent of frequency but proportional to the relaxation exponent:31 δ ) nπ/2 or tan δ ) G′′/G′ ) tan(nπ/2)
(4)
These results suggest that the following scaling relations describes the incipient gel: G′(ω) ∝ G′′(ω) ∝ ωn
(5)
However, when the dynamic moduli exhibit very weak frequency dependence, it may be advantageously to monitor the evolution of viscoelastic properties during gelation
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Figure 7. Characteristics of incipient gels of the pectin/methanol/ water system. Effects of methanol on the gelation time (a), on the viscoelastic exponent n (b) (see eq 4), on the gel strength parameter S (c) (calculated from eq 2), and on the fractal dimension df (d) (calculated from eq 9).
Figure 6. Frequency dependence of the complex viscosity (log-log plot) at different stages during the gelation process for the pectin/ methanol/water system at different methanol/water compositions.
through the complex viscosity |η*(ω)| (see eq 2). In an analogous way as for the dynamic moduli, we may at the gel point portray the frequency dependence of the complex viscosity in terms of a power law32 |η*(ω)| ) aSωm
(6)
m≡n-1
(7)
with
and a)
π Γ(n) sin(nπ)
(8)
where m is the complex viscosity relaxation exponent of the incipient network. It is clear that if the frequency dependence of the dynamic moduli is weak (n is close to 0), a strong, easily detected, dependence of m (m approaches -1) is observed. Values of m close to zero announce liquidlike behavior, whereas values of m approaching -1 suggest solidlike response of the system. The frequency dependence of the complex viscosity, as measured in small amplitude oscillatory shear experiments, at different stages (where ) (t - tg)/tg, t is the considered time and tg is the time of gelation, is the relative distance from the gel point) during the gelation process for the pectin/ methanol/water system at various concentrations of methanol is displayed in Figure 6. It is interesting to note that power laws in frequency are observed, not only at the gel point ( ) 0), but also in the pregel ( < 0) and postgel ( > 0) domain. It is only for the pectin/water system that there is a clear trend toward an enhanced elastic response (the value
of m increases) as the gel evolves, whereas for the pectin/ methanol/water mixtures, this trend cannot be established, which indicates that extended oscillatory shear perturbation on the system does not further strengthened the solidlike behavior of the gel. The results at the gel point suggest that the most solidlike gel is formed in the presence of moderate concentrations of methanol. High vales of m have previously been reported10 from a rheological study of temperatureinduced gelation of pectin-chitosan mixtures. The linear viscoelastic characteristics of incipient shearinduced gels of pectin in mixtures of methanol/water of various compositions are illustrated in Figure 7. At low and moderate methanol concentrations, the gelation time seems to exhibit an irregular behavior (Figure 7a). The reason for this trend is not clear, but we should bear in mind that, despite a careful aspiration to establish a unitary procedure for the rheological experiments, it is difficult to guarantee solutions that are extremely sensitive to shear perturbations to have experienced exactly the same conditions. However, at higher methanol concentrations, the gelation time becomes significantly longer, which probably can be traced to the proximity of phase separation and slowing down of the gelation process as a result of structural heterogeneities. The power law exponent n falls off with the percentage of methanol up to 6 wt %, suggesting that “harder” incipient gels are formed under these conditions. At higher concentrations of methanol, phase separation is approached, and this seems to promote the formation of “softer” gels. The finding that the dynamic moduli for incipient gels are nearly frequency independent has previously been reported10,11,33-35 for other polysaccharide systems that have not been exposed to shear-induced gelation. The gel strength parameter S raises with increasing methanol content in the mixture, and this feature probably reflects that both the cross-linking density and the chain rigidity increase. Because of deteriorated thermodynamic conditions, enhanced intermolecular chain associations favor an augmented cross-link density and probably also the chain rigidity. Muthukumar developed a theoretical model,36 based on the assumption that variations in the strand length between
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than their rate of reformation, resulting in a decrease in the junction density and hence a drop in the viscosity. The inset plot illustrates the change of the position of the maximum (shear ratemax), with respect to shear rate, of the flow curve as the methanol concentration of the mixture increases. Up to 6 wt % of methanol, the maximum is shifted toward higher shear rate values, which may announce that the disruption of the network is made more difficult. Shear-induced gelation of gelatin systems has recently been reported.40 Conclusions
Figure 8. Shear rate dependence of the viscosity for the pectin/ methanol/water system at different methanol/water compositions. Data obtained under increasing shear rate have been collected. The inset plot shows how the position of the maximum, with respect to shear rate, of the shear curves changes with methanol concentration.
cross-linking points of the network give rise to changes of the excluded volume interactions, to rationalize values of the exponent n in the whole physically accessible range (0 < n
