Biomacromolecules 2002, 3, 466-474
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Interpenetrating Network Formation in Agarose-K-Carrageenan Gel Composites Eleonora Amici, Allan H. Clark,* Valery Normand,† and Nick B. Johnson Unilever R&D Colworth, Colworth Laboratory, Sharnbrook, Bedford MK44 1LQ, U.K. Received November 5, 2001; Revised Manuscript Received February 11, 2002
Thermal, mechanical, turbidity, and microscope evidence is provided which strongly suggests molecular interpenetrating network (IPN) formation by mixtures of the seaweed polysaccharides agarose and κ-carrageenan. Over a range of ionic strength, and potassium content, there is no evidence for synergistic coupling of the networks, and simple phase separation (demixing) can definitely be ruled out. At low ionic strength, where the agarose gels first, differential scanning calorimetry evidence shows some influence of the carrageenan on the agarose ordering enthalpy, particularly at higher polymer concentrations. As the potassium level is increased, however, and the order of gelling is reversed, this effect disappears. Cure behavior for the systems at high ionic strength can be described as a simple summation of the pure component contributions. At low ionic strength, on the other hand, the modulus behavior is more complex, suggesting either a modification, in the mixture, of the κ-carrageenan gelling parameters or a more complex modulus additivity rule. Introduction Gelling of ternary aqueous biopolymer mixtures (two polymers, one solvent) has been studied for many years, but it is only recently that the full subtleties of this phenomenon have begun to be appreciated. An early view1-3 was that dissimilar biopolymers in a mixed solution were likely to be highly incompatible, particularly if gelation was also involved with its concomitant increase in molecular weight. Mixed gels based on biopolymer pairs were therefore seen as substantially segregated and were expected to take the form of suspensions of gelled droplets of one component in a gelled matrix of the other. In many cases this was true, although additional features such as a droplet-within-droplet microstructure (secondary demixing), or phase bicontinuity close to the point of phase inversion, were soon also recognized. Mathematical models3 relating shear moduli to composition were derived, which assumed such morphology, and regarded the gelling polymers as existing in their own separate compartments. These models were based on the idea of an “effective” biopolymer concentration (i.e., the real local concentration within the compartment) as opposed to the “nominal” or starting (overall average) solution concentration. Research during the past few years4-10 has demonstrated, however, that the formation of mixed biopolymer gels can be a much more varied and complex process than envisaged by these early models. Awareness of this has steadily emerged from work on the kinetics of mixed gel formation using techniques such as time-resolved small-angle light scattering, turbidity measurements, cure rheology, and microscopy. This has taken advantage of the fact that the † Present address: Firmenich SA, 1, Route des Jeunes, CH-1211 Geneva 8, Switzerland.
binodals of ternary biopolymer solutions rarely extend fully to the composition axes, i.e., that the degree of demixing in such ternary systems is rarely complete prior to gelation, and that the binodal can move quite considerably with temperature. This allows temperature quenches to be performed on homogeneous starting solutions, as opposed to preformed water-in-water emulsions. When this is done, gelation can be arranged to compete more equally with the demixing process, and more complex microstructural outcomes, than the simple droplet form, become possible. Trapped spinodal structures4,5 are generated, for example, analogous to those often reported for binary synthetic polymer blends. As the competition between demixing and gelling moves more and more in favor of the latter, a limiting form of behavour can emerge in which even the concentration fluctuations of the spinodal structure disappear. This has been illustrated in a recent study11 by the present authors on the gellan-agarose system in which a so-called molecularly interpenetrating network (IPN) was seen to form; i.e., one polysaccharide gel network appeared to pass through the pore structure of the other. There was no evidence of interaction between these networks nor was there microscopic evidence of any kind of physical phase separation. Such IPN formation (which should be distinguished from interpenetrating phase formation, or phase bicontinuity) was considered to arise mainly because of the intrinsically low tendency of a charged-uncharged polysaccharide mixture to phase separate (counterion entropy effect12). Fully phase-separated systems and IPNs represent extremes of behavior and are significantly different. Modeling the mechanical properties of IPNs, for example, raises new issues when theoretical description is attempted.11 Although the idea of an effective concentration can be retained, this no longer
10.1021/bm010157z CCC: $22.00 © 2002 American Chemical Society Published on Web 03/15/2002
Network Formation in Gel Composites
has the simple meaning of a real concentration effect. For the gellan-agarose system, for example, the overall composite gel modulus was found to be very nearly equal to the sum of the values expected for the two component networks in isolation (that is, at their nominal concentrations). There was no evidence of the mutual concentration expected for phase-separated systems. There was a special subtlety, however. Close inspection of the relevant cure data showed that the apparent simple additivity was not perfect, the gellan, in particular, being influenced in its gelling behavior by the agarose coil molecules surrounding it. A model was proposed for this situation which attempted to retain the gelling properties of the pure components as a reference (as had been done in previous treatments of genuinely phaseseparated composites). In this case, however, the model operated in terms of changes in biopolymer gelling parameters (cross-linking rate constants, etc.) accompanying mixed gel formation, rather than real changes in concentration. The present paper continues this exploration of molecular IPN formation by examining the agarose-κ-carrageenan system. This again is an example of an uncharged-charged polysaccharide combination, and previous work by Zhang and Rochas13 on this system has suggested independent gelation of the components. The analogy with agarosegellan is therefore strong, and a parallel, and more in-depth, study of agarose and κ-carrageenan seems worthwhile. The previous investigation of agarose-κ-carrageenan is therefore extended in the present publication, in terms of the range of techniques applied, and the range of ionic strength and counterion conditions considered. Particular attention is given to the applicability of models previously developed11 to treat network formation by agarose and gellan. Agarose and κ-carrageenan are of course well-known seaweed polysaccharides,14 and in the present case, the carrageenan used was essentially a sodium salt form with low gelling capacity. In the experiments to be described, however, this capacity was progressively enhanced by addition of increasing levels of potassium ions. This raised the gelation temperature of the carrageenan, and its gel strength at a given concentration, and made it possible to move from a situation in which the agarose gelled first, to the reverse, where the carrageenan was the first gelling biopolymer. As in the previous work11 on agarose-gellan, the behavior of the new system was studied by a combination of physical techniques including dynamic mechanical spectroscopy, large deformation mechanical testing, differential scanning calorimetry (DSC), turbidity measurements, and transmission electron microscopy (TEM). Materials and Methods Materials. Agarose (a major constituent of agar) originates in red seaweeds (Rhodophyceae) and is a linear uncharged polymer with an idealized structure based on a disaccharide repeat unit consisting of (1-3)-linked β-D-galactose and (14)-linked 3,6-anhydro-R-L-galactose. It forms cold-set gels based on aggregates of ordered double helices. The sample used here was purchased from Sigma (type 1-A, low EEO, A0169). The sulfate content was less than 0.2% w/w. The
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molecular mass distribution was determined in the coil form at 60 °C using size exclusion chromatography and light scattering. The light scattering experiments employed a DAWN-F MALLS photometer (Wyatt Technology, USA) equipped with a He-Ne laser (633 nm, 5 mW). Molecular weight averages were Mw ) 1.7 × 105 and Mn ) 1.0 × 105. κ-Carrageenan is a sulfated galactan polymer which can be extracted from various species of red seaweed (e.g., Eucheuma spinosum). It has an idealized primary chain structure based on the repeating disaccharide (1-3)-linked β-D-galactose and (1-4)-linked 3,6-anhydro-D-galactose, the β-D-galactose being sulfated at the 4 position. κ-Carrageenan, like agarose, also forms cold set gels through double helix formation, particularly when in the potassium form, i.e., its gelation temperature and gel strength are strongly influenced by ionic strength and by counterion identity. In the present case the κ-carrageenan sample was supplied by SKW as a mainly sodium salt form. Its molecular weight distribution was measured as described for agarose, the principal averages being Mw ) 7.3 × 105 and Mn ) 4.4 × 105. Solutions were prepared by accurate weighing of components at ambient, followed by heating to boiling point for 25 min with stirring. Both sodium chloride and potassium chloride were added as supporting electrolyte, the latter to strongly influence the carrageenan gelation. A strategy was adopted for each potassium level studied of keeping the ionic strength I of the initial coil solution constant as polymer concentration was varied. To achieve this, the formula I ) Cs + 0.5Ccp suggested in previous literature15 was adopted, with the sodium chloride level being adjusted to compensate for changing amounts of sodium ions from the carrageenan polymer. Here Cs is the molar concentration of the supporting 1:1 electrolyte in the solvent, and Ccp is the molar concentration of counterions from the polyelectrolyte. This did not ensure constancy of the ionic strength throughout gelling, of course, but as a practical systematic procedure, it did lead to certain simplifications in the cure experiments (see later). Small-Deformation Rheology. Measurements of storage and loss shear moduli (G′, G′′) were performed on a Carrimed CSL 500 stress-controlled rheometer using a coaxial cylinder geometry (internal radius 20 mm, external 23 mm, gap 3 mm). Hot solutions were introduced between the cylinders. They were supported at the bottom by a layer of perfluorodecaline and sealed at the top with silicone oil. Experiments were performed at a constant frequency of 1 Hz and at 0.5% strain. Temperature control was by means of a programmable circulating water bath. Solutions were loaded at 60 °C, on to the rheometer, cooled to 10 °C at 1 °C/min, held at 10 °C for 3 h, and then finally heated to 85 °C (1 °C/min). Large-Deformation Tensile Testing. Measurements of the tensile strengths of the gels were performed at 10 °C, under uniaxial testing, on an Instron Universal testing machine (model 4502). Test parameters were as follows: load cell 10 kN, cross head speed 100 mm/min, and sampling rate 25 points/s. Hot solutions (60 °C) were poured between parallel glass plates (spaced at 1.4 mm) immersed in a programmable water bath, and then cooled to 10 °C at 1 °C/min. The plates were stored at 5 °C for 20 h and then
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equilibrated within the Instron cabinet at 10 °C for 2 h. Tensile test pieces (60 mm gauge length, 6 mm width) were cut from the gel sheets using a “dog bone” shaped cutter and gripped within the Instron test frame using filter paper and attached cardboard tabs. Between 8 and 10 replicates were tested for each composition considered. Corrected true stress and strain were calculated as F(Lo + ∆L)/(AoLo) and ln((Lo + ∆L)/Lo, where L was the varying length of the test region of the sample, Lo its original height, Ao its original cross-section, and F the applied load. The true failure stress and strain were recorded for each sample, and tensile elastic moduli were calculated from the initial linear parts of the stress-strain curves in the usual way. Differential Scanning Calorimetry. Measurements were performed on a Setaram micro-DSC III batch and flow calorimeter. Sample pans contained accurately measured amounts of sample (close to 800 mg). NaCl solutions having the same ionic strength as the samples were used as references. Before each measurement, materials were heated to high temperature to eliminate thermal history effects, then cooled, and heated again at 1 °C/min. Enthalpies of transitions, onset temperatures, and peak maximum temperatures were calculated using Setaram software, which carried out baseline subtraction and integration steps. Turbidity Measurements. These were performed on a Perkin-Elmer UV-vis scanning spectrophotometer in the wavelength range 400-800 nm and for a path length of 1 cm. Solutions and reference samples (NaCl + KCl at appropriate ionic strength) were filtered (Whatman PUDF filter, 0.45 µm) and added to preheated quartz cuvettes. These were equilibrated at 60 °C, cooled to 10 °C at 1 °C/min, and held at 10 oC for 1 h. Temperature was controlled using a Peltier device. Transmission Electron Microscopy. Vials containing hot solutions were cooled from 60 to 10 °C at 1 °C/min and stored at 4 °C. The gelled samples were cut into 1 mm cubes and immersed in 0.05% ruthenium tetroxide at this same temperature for 2 h. The fixed samples were then washed in distilled water and progressively dehydrated using aqueous ethanol solutions of increasing concentration (30 min in 50, 70, 90, and 100% solutions). The samples were then resin embedded in LR White/GMA (7:3) for 8 days and polymerized at 55 °C in an oven overnight. Sectioning was performed using an Ultracut E microtome and, in some experiments, sections were antibody labeled using rabbit antiagar or anti-κ-carrageenan, as appropriate. The rabbit antibody labeled sections were further treated with goat antirabbit gold conjugates. All sections were counterstained with uranyl acetate (10 min) and lead citrate (30 s). Gold conjugates were purchased from British BioCell International, and the antibodies were raised at the Royal Holloway Hospital, U.K. Micrographs were recorded using a JEOL 1220 TEM. Results Cure Curve Studies. The cure behavior of the agarose used in this study has been described in detail in the previous article11 on gellan-agarose systems. There it was shown that
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Figure 1. Gelation of κ-carrageenan at I ) 79 mM (Na+ ) 65 mM; K+ ) 14 mM) and in the concentration range 0.5% w/w to 2.75% w/w. Small deformation oscillatory measurements (1 Hz, 0.5% strain) provide the variation of G′ with time. Solutions were cooled from 60 to 10 °C at 1 °C/min and held at 10 °C.
for agarose concentration in the range 0.25% w/w to 1.75% w/w, cure curves had a similar shape in log-log form and could be superimposed to provide a master curve. The critical gelling concentration was estimated to be 0.07 ( 0.01% w/w, and the long time limiting modulus values showed a power law dependence on concentration (exponent 2.7) over the limited concentration range studied. On melting the gels by heating to 85 °C at 1 °C/min, the modulus began a significant fall at 20 °C, but the mechanical response was still gel-like (G′ > G′′) at the highest temperature accessed. The cure behavior of the κ-carrageenan was studied over a range of ionic strength and potassium concentration. At low ionic strength I ) 79 mM, and in conditions of counterion content (Na+ ) 65 mM; K+ ) 14 mM), cure curves were obtained as shown in Figure 1. As in the agarose case, these could easily be superimposed into master curve form by appropriate shifts along the modulus (log display) axis in relation to one particular data set chosen as a reference (reference concentration). No such shifts were required along the log time axis indicating a very low sensitivity of the apparent gel time to concentration variation, at least over the concentration range (0.5-2.75% w/w) considered, and for the kinetic conditions of cooling. This probably reflects both the rapid rate of gelation which prevails for κ-carrageenan once the gelation temperature is reached on the downward temperature scan and the lack of concentration dependence of the initial ordering temperature (see later section on DSC results). Increasing the potassium level at constant sodium concentration (fixed at 65 mM) led to an increase in gelation temperature for the carrageenan and an increase in the long time limiting modulus for a given polymer concentration. An interesting additional feature was the emergence in the cure curve of what seemed to be evidence for a two-stage gelation process (i.e., presence in the cure curve of two distinguishable steps). This is shown in Figure 2 for the particular case of a κ-carrageenan concentration of 1.25% w/w. As mentioned earlier, these changes, induced by addition of potassium, allowed a changeover in terms of the identity of the first gelling polymer component in the mixtures, i.e., from the agarose to the carrageenan.
Network Formation in Gel Composites
Figure 2. Gelation of κ-carrageenan (1.25% w/w) at different ionic strengths (Na+ ) 65 mM; K+ from 14 to 69 mM). Small deformation oscillatory measurements (1 Hz, 0.5% strain) show the variation of G′ with time. Solutions were cooled from 60 to 10 °C at 1 °C/min and held at 10 °C.
Figure 3. Gelation of agarose, and agarose-κ-carrageenan mixtures at I ) 79 mM (Na+ ) 65 mM; K+ ) 14 mM). Small deformation oscillatory measurements (1 Hz, 0.5% strain) provide the variation of G′ with time. Solutions were cooled from 60 to 10 °C at 1 °C/min and held at 10 °C for 2 h.
Interestingly, the gelation of κ-carrageenan in a mixed ion situation (actually NaI, CsI) has been described elsewhere in the literature,16 including the two-step character introduced into the cure curve profiles. In this previous work, the twostage gelation is described as kinetic, since it seems to disappear on reducing the cooling rate. In general, potassium ions promote ordering of κ-carrageenan, and aggregation of the double helices, through some form of specific ion binding to the ordered form. It is therefore tempting to describe the first phase of curing seen here as due to formation of double helices and the remainder to helix aggregation, but there is no direct evidence to support this. It is interesting that despite the two-step nature of the cure curves induced by adding potassium, master curve superposition of these could still be achieved, provided that the ionic strength (and especially the potassium level) was fixed, over the concentration range of interest. Curves for samples having different potassium levels could not be combined in this way. The cure behavior of mixtures of agarose and κ-carrageenan at the lowest ionic strength (I ) 79 mM) and lowest potassium level (14 mM) are typified by the results shown in Figure 3 for a particular concentration of agarose (0.625% w/w) and increasing carrageenan. Here the initial period of
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Figure 4. Melting of gelled agarose, and agarose-κ-carrageenan mixtures, under same electrolyte conditions as Figure 3. Small deformation oscillatory measurements (1 Hz, 0.5% strain) provide the variation of G′ with temperature. Solutions were heated from 10 to 85 °C at 1 °C/min.
modulus increase (shoulder in Figure 3) can be identified (see later DSC results) with the agarose component and the second with the carrageenan. When these composite gels are reheated, the behavior shown in Figure 4 (this time for a 0.375% w/w agarose series) was typically found, with the κ-carrageenan providing the first phase of melting and the agarose the second. In general, at low ionic strength, and low potassium concentration, the agarose melting profile was only slightly influenced in the composites, since melting of the pure agarose at the same nominal concentration provided a reasonable fit to the high-temperature melting data for the mixed gels. Subtracting this last from the full composite meltdown data allowed an estimate to be made of the κ-carrageenan contribution to the composite modulus at 10 °C, though this depended on the assumption of simple additivity of the component moduli (see later Discussion). A similar ambiguity was recognized on attempting to analyze the cure data. Here again, straightforward and unequivocal resolution of the initial cooling cure curve into contributions from the two-component polymers, as was achieved in the previous gellan-agarose study, was not possible in the current situation, owing to much greater overlap of the gelling events. It was certain, however, that, for whatever reason (complex modulus additivity and/or changes in gelling characteristics) neither the cure curves nor the melting curves, at low potassium content, could be accurately reproduced by simple addition of pure component contributions at the nominal concentrations. On changing to increasingly higher levels of potassium, and higher total ionic strength, the cure behavior of the agarose-κ-carrageenan mixtures showed a change from the behavior of Figure 3 (lowest ionic strength) to that presented in Figure 5 (highest ionic strength I ) 134 mM; Na+ ) 65 mM; K+ ) 69 mM). In this situation (agarose 1.25% w/w, carrageenan 2.0% w/w) the period of agarose gelation clearly lies between the two steps that are features of the high potassium carrageenan contribution. The κ-carrageenan now starts the gelation process but, at the cooling rate involved, it also ends it, the agarose phase of aggregation, being “bracketed” by the contribution from the charged polysaccharide. The level of added potassium at which the cross
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Figure 5. Gelation of agarose, κ-carrageenan, and an agarose-κcarrageenan mixture at I ) 134 mM (Na+ ) 65 mM; K+ ) 69 mM). Small deformation oscillatory measurements (1 Hz, 0.5% strain) provide the variation of G′ with time. Solutions were cooled from 60 to 10 °C at 1 °C/min and held at 10 °C for 3 h.
Figure 6. Melting of agarose and agarose-κ-carrageenan mixtures over a range of ionic strengths and potassium levels (14-69 mM). Small deformation oscillatory measurements (1 Hz, 0.5% strain) provide the variation of G′ with temperature. Solutions were heated from 10 to 85 °C at 1 °C/min.
over from agarose gelling first to carrageenan gelling first takes place is around 36 mM (I ) 101 mM). Interestingly, the cure data obtained at the higher ionic strength and potassium levels could be closely represented by sums of contributions from the components at the same nominal system concentrations. However, the melt down curves shown in Figure 6 for an agarose-κ-carrageenan system (agarose 0.75% w/w, carrageenan 1.25% w/w) at increasing potassium levels, warn that, in the composite gels, the agarose melting contribution is not generally identical to that expected for the pure polysaccharide. The simple additivity result at high potassium levels may therefore be fortuitous and should be treated with care. Large-Deformation Tensile Measurements. Stressstrain results for agarose (1.25% w/w) and a series of systems containing increasing κ-carrageenan levels, but at the same low ionic strength (I ) 79 mM) and low potassium content (14 mM), appear in Figure 7. The basic character of the agarose brittle failure is retained by the composites. The stress-to-break increases as the κ-carrageenan is added, with the strain-to-break remaining approximately constant. The individual stress-strain curves all tend to be concave upward
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Figure 7. Representative stress-strain curves for agarose and agarose-κ-carrageenan gels (I ) 79 mM; Na+ ) 65 mM; K+ ) 14 mM). Tension rate was 100 mm/min.
Figure 8. DSC heating (open symbols) and cooling (solid symbols) scans of κ-carrageenan solutions/gels at 0.5% (squares), 1.25% (triangles), and 2% (circles) (w/w). Scan rate 1 °C/min. Electrolyte conditions as for Figure 7.
and divide roughly into two linear regions, the second being steeper than the first, and indicating strain hardening. Poisson’s ratio was around 0.4 for all of these gels. Very similar results were obtained for the composites at higher potassium content. The closely similar curved shapes observed for all of the gels studied, and the constancy of the strain-to-break, were taken as support for a molecular IPN description. Differential Scanning Calorimetry. DSC scans (both cooling and heating) have already been discussed for the agarose component as part of the previous work11 on gellanagarose. In general, for agarose, the cooling scans show a fairly sharp exotherm starting at around 40 °C, but with some variation of this temperature with concentration. The heating scans are different, in that they show a continuous melting endotherm extending up to 90 °C and beyond, and indicating progressive melting of aggregates of helices over a wide temperature range. This hysteresis property is well-known for polysaccharides gelling through aggregation of ordered helices. Corresponding results for κ-carrageenan sols and gels at three concentrations appear in Figure 8. On cooling, ordering occurs around 25 °C for the low ionic strength conditions employed (I ) 79 mM; Na+ ) 65 mM; K+ ) 14 mM) and shows little polymer concentration dependence. A hysteresis
Network Formation in Gel Composites
Figure 9. DSC heating (open symbols, broken line) and cooling (solid symbols, solid line) scans of agarose 0.625% (lines), κ-carrageenan 1.25% (squares), and agarose 0.625%-κ-carrageenan 1.25% (triangles) (w/w) solutions/gels. Scan rate 1 °C/min. Electrolyte conditions as for Figure 7.
Figure 10. Enthalpies of setting for agarose, κ-carrageenan, and their mixtures for three concentration series (fixed agarose, increasing carrageenan). The cross-centered symbols refer to the sums of the enthalpy of the agarose in the series, and the enthalpy of the κ-carrageenan at the appropriate concentration. Other symbols are as shown.
effect is again observed on heating, but this is much smaller than that found for agarose, suggesting a more limited helix aggregation event and/or a much lower aggregate melting temperature. Turbidity data, to be discussed below, would seem to support the former explanation. Results for a typical agarose-κ-carrageenan mixture under the same salt conditions appear in Figure 9. There, the cooling curve shows two exotherms, and from the previous figures, these can be identified as corresponding to the agarose component gelling first and then the carrageenan. Close examination of onset and peak temperatures and the integrated enthalpies of the peaks shows that these agree well with expectations based on the behavior of the pure components at the same concentrations. This simple result breaks down only at the highest carrageenan and agarose levels where the enthalpy, in particular, is smaller in absolute magnitude than would be expected from simple additivity (Figure 10). Modeling exercises applied to the data strongly suggest that it is the agarose component that is affected, not the carrageenan. The agarose, it appears, is less able to form perfect helices (or forms less helix) in the presence of a
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Figure 11. DSC cooling scans of agarose 0.75% (w/w)-κ-carrageenan 1.25% (w/w) at different ionic strengths (I ) 79 mM circles, I ) 101 mM stars, I ) 134 mM squares). For potassium levels, subtract Na+ ) 65 mM. Scan rate 1 °C/min.
higher carrageenan concentration than normal. This conclusion is reached, however, on the assumption that the measured enthalpy derives purely from the helix formation (often made for agarose gelation but perhaps not conclusively proved). Finally, Figure 11 shows how DSC cooling scans for the mixtures change as potassium content is increased. This is in keeping with the idea of a changeover in the order of gelation from agarose first to carrageenan first. Interestingly, at high ionic strength, and high potassium levels, it is found that the peak temperatures and the integrated enthalpies agree very closely with what would be expected for the pure components ordering independently. The agarose enthalpy anomaly observed at low ionic strength now disappears, a result that seems to agree well with the small-deformation cure data reported earlier. It agrees less well, perhaps, with the modulus-temperature melting data also discussed. However, it should be remembered that melting data of this kind are more likely to reflect the distribution of aggregate size than the extent and perfection of individual helix formation. Turbidity Measurements. Figure 12 shows the evolution of the turbidity (measured at 400 nm) on cooling agarose, κ-carrageenan, and mixtures of these, from 60 to 10 °C at 1 °C/min. These systems were studied at the lowest ionic strength (I ) 79 mM) and potassium level (K+ ) 14 mM). The pure agarose system has a much higher turbidity than the pure carrageenan probably because its gel fibers are much thicker (see the above DSC arguement about hysteresis and helix aggregation). It is clear too that under the prevailing conditions the pure agarose begins to order, and aggregate, earlier than the pure carrageenan. For the mixture, the turbidity is always lower than that of the pure agarose, and this is particularly obvious at longer times, after the carrageenan has gelled. When this latter process starts, as Figure 12 shows, the agarose turbidity increase becomes arrested quite dramatically, though a small increase is still seen, corresponding to development of the carrageenan network. As for the previous gellan-agarose system, turbidity values are clearly depressed during gelation, and this seems
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Figure 14. TEM micrograph of κ-carrageenan 2% w/w gel at I ) 134 mM (Na+ ) 65 mM; K+ ) 69 mM). Image width was 3.6 µm. Figure 12. Evolution of turbidity on cooling agarose (1.25% w/w), κ-carrageenan (2.0% w/w), and their mixture, from 60 to 10 °C at 1 °C/min. I ) 79 mM (Na+ ) 65 mM; K+ ) 14 mM).
Figure 15. TEM micrograph of agarose 1.25% (w/w)-κ-carrageenan 2% (w/w) gel. Image width was 3.6 µm. Electrolyte conditions as for Figure 14. Figure 13. TEM micrograph of agarose 1.25% w/w gel. Image width 3.6 µm.
to be characteristic of molecular IPN formation by chargeuncharged polysaccharide pairs. This may indicate interference between scattering from the two networks when they form and intertwine or, perhaps equivalently, a fall in pore size, when the second component orders within the first. At any rate, there is no sign of the increase in turbidity expected if simple liquid-liquid demixing were part of the gelation process. Very similar results were found for these mixtures at higher potassium levels, and similar conclusions were reached. Microscopy. As for the gellan-agarose system,11 examination of the initially hot mixtures by optical microscopy gave no indication of simple phase separation (i.e., formation of water-in-water emulsions). For the highest ionic strength systems studied (I ) 134 mM; Na+ ) 65 mM; K+ ) 69 mM), transmission electron micrographs are presented for pure agarose (1.25% w/w), pure κ-carrageenan (2.0% w/w), and their mixture, in Figures 13, 14, and 15, respectively. In these images only conventional counterstaining has been used to achieve contrast. The agarose network appearing in Figure 13 is much as described in previous publications (e.g., ref 11), and takes the form of a fairly uniform array of long thick fibers. The κ-carrageenan network (Figure 14) is different, however, in
that it is more heterogeneous (network density fluctuations) and the fibers forming it seem thinner and more frequently branched. The more heterogeneous character of the carrageenan network than that of agarose is perhaps surprising in view of the much lower turbidity of the carrageenan gel (see previous section) though this could be explained if gel turbidity is as strongly influenced by fiber diameter, as network uniformity. There is also the fact that previous electron microscope work17 has suggested a fairly uniform distribution of network fibers for the potassium κ-carrageenan system. Interestingly, in this last work, fine fibers, forming a transient structure, develop into straighter and thicker fibers. There may be an element of this in Figure 14, but in view of the fact that the two investigations were performed with different samples, under different salt conditions, and using different microscope methods, direct comparison is probably unwise. It is not impossible, however, that the embedding methods used here have had some influence on the final network appearance. The electron micrograph for the mixed gel (Figure 15), made at the highest ionic strength and potassium level, appears to be close to a superposition of the images obtained for the pure components. Even with conventional staining it is possible to distinguish the agarose and carrageenan contributions and to confirm that these networks are quite intimately mixed. The simplest interpretation of these results
Network Formation in Gel Composites
is that, at the potassium level involved, the carrageenan forms its network first, in a manner close to that characteristic of the pure polymer gelation. The agarose then gels in its normal manner within the pores of this network. Separate studies of the mixtures using agarose antibody staining tended to support this view, though one could not be absolutely sure that the fiber structure of the agarose was completely unperturbed by the carrageenan. Micrographs (not presented here) were also obtained for the mixed systems at the lowest ionic strength and potassium level. The κ-carrageenan network was more difficult to stain and visualize by conventional methods in these experiments, but specific carrageenan antibody labeling helped to identify its presence within the pores of the agarose. The mixed gel structure did not seem to be fundamentally altered by the changing salt conditions. Discussion As in the previous gellan-agarose example,11 substantial phase separation of the components in the present agaroseκ-carrageenan mixtures can certainly be excluded. A process of only limited microphase separation, during gelation, has to be considered more seriously, however. A small degree of this appears to be indicated in the micrograph shown in Figure 15 for the high ionic strength, high potassium, mixture. Had the network density fluctuations seen there not also been a feature of the gelation of the pure carrageenan component (Figure 14), one might have been tempted to propose limited segregation of the two polymers prior to, or during, gelation. What actually seems to be occurring is gelation of the carrageenan in a way that retains its normal native degree of density fluctuation under the prevailing salt conditions. Supporting evidence for this interpretation is the absence of any sign of induced agarose density fluctuations in Figure 15 or, perhaps more particularly, a similar absence in composite micrographs, where the agarose antibody was specifically employed (not shown here). Although all the evidence described here points to IPN formation for the various agarose-carrageenan systems examined and supports the conclusions of the earlier study13 by Zhang and Rochas, close quantitative analysis warns that it would be a mistake to equate this with complete independence of behavior of the two biopolymer components. Thus, at low ionic strength, and low potassium, and for mixtures at the highest total polysaccharide content, the agarose ordering enthalpy was found to be reduced. At higher ionic strengths, the DSC results were certainly simpler (more additive), but rheological studies of the melt down suggested agarose anomalies. In terms of the cure behavior, and the low temperature shear modulus, the picture is also more complicated, especially at low ionic strength. Here, neither the cure curves nor the melt down data can be accurately described as sums of pure component contributions at the nominal concentrations. Either the idea of simply adding these contributions (for an IPN) is inappropriate (perfectly possible) or the gelling parameters of the components (e.g., cross-linking rate constants) are influenced on mixing or both.
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For the present system it is difficult to separate these effects convincingly. The high-temperature agarose contribution to the rheological melting of the low ionic strength gels discussed earlier certainly points to the agarose behaving as it would in isolation at the same nominal concentration. This is despite the DSC anomalies found for this component (see Figure 10). There is, however, considerable overlap of the agarose melting curve with that of the carrageenan at the lowest temperatures. One cannot be certain, therefore, that the agarose contribution is identical to its pure behavior in this region, though this seems likely. We might therefore conclude that in the low ionic strength, low potassium, situation, the agarose is relatively unaffected (certainly in rheological terms) by the carrageenan presence. The situation is rather more complicated for the κ-carrageenan in these mixtures. If, at low ionic strength, the agarose contribution (at nominal concentration) is simply subtracted from the melt-down data, or indeed from the initial cure data, neither the nominal carrageenan cure nor the nominal melt down response is obtained. Over a considerable range of agarose and carrageenan concentrations, the carrageenan behaves, in fact, as if it had been concentrated up. This is very similar to conclusions reached11,18 about the behavior of gellan in the presence of either agarose or Paselli SA2. There, however, the results were not subject to the assumption of simple modulus additivity and were more clear-cut. The apparent concentration of the gellan found in the previous work11,18 was interpreted as indicating a significant change in gelling parameters for the gellan in the mixed water-agarose or water-maltodextrin solvent, rather than any real concentration effect (i.e., real compartmentalization). The apparent concentration of the carrageenan in the present low ionic strength mixtures could be interpreted similarly but, in the present case, an alternative explanation based on more complex additivity of the network contributions to the modulus cannot be excluded. The matter therefore remains open to doubt. Melting behavior for the agarose-κ-carrageenan mixtures at high ionic strength is more complex than that seen at the lowest value just discussed. The agarose contribution, at high temperature, was found to be unlike that of pure agarose, in most of the high potassium mixtures examined. The agarose network seems to have been somewhat modified by the carrageenan, particularly in terms of the degree of helix aggregation involved. The helix formation, itself, appears to have been less affected, as the DSC results were found to be additive in terms of the pure component contributions. The overall modification of the agarose gelling may not be severe, however, as the cure data for the high ionic strength systems can be well described by simple summation of the nominal component contributions. Caution is advisable, however. The simple summation found could arise from a cancellation of effects (network additivity and changes to gelling parameters) and be largely accidental. Despite the above uncertainties regarding the detailed quantitative description of IPN formation and behavior, the basic features of the phenomenon seem to have been established, both by the present work and by the previous studies11,18 of gellan mixed with agarose and with a modified
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starch. An extreme of mixed gel formation is involved, in which segregation of the network-forming species is at a minimum. The effect is promoted by combining charged and uncharged polymers and by fast network formation by at least one of the components. Mechanically speaking, the gels show very similar large deformation and failure properties to the pure systems from which they are formed, but the stress-to-break is usually increased. Modeling the mechanical properties of these systems using the gelling behaviors of their individual biopolymer components as reference inputs is significantly less well founded than for their phaseseparated counterparts. Not only are there quite severe theoretical difficulties in doing this, but experimental testing of such models is frustrated by the difficulty of measuring the intrinsic cross-linking kinetics of the components individually, and so separating these from issues of network property additivity. Overcoming these problems will be a formidable challenge for the future. Acknowledgment. The authors thank colleagues at the Colworth Laboratory, Professor E. R. Morris, Cork University, and Dr. R. K. Richardson, Silsoe College, Cranfield University, for many helpful discussions of polysaccharide gelation and the implications of IPN network formation. They also thank Miss A. Russell for molecular weight determination. References and Notes (1) Clark, A. H.; Richardson, R. K.; Robinson, G.; Ross-Murphy, S. B.; Weaver, A. C. Prog. Food Nutr. Sci. 1982, 6, 149-160. (2) Clark, A. H.; Richardson, R. K.; Ross-Murphy, S. B.; Stubbs, J. M. Macromolecules 1983, 16, 1367-1374.
Amici et al. (3) Clark, A. H. In Food Structure and BehaViour; Lillford, P. J., Blanshard, J. M. V., Eds.; Academic Press: London, 1987; pp 1334. (4) Clark, A. H. In Biopolymer Mixtures; Harding, S. E., Hill, S. E., Mitchell, J. R., Eds.; Nottingham University Press: Nottingham, 1995; pp 37-64. (5) Tromp, R. H.; Rennie, A. R.; Jones, R. A. L. Macromolecules 1995, 28, 4129-4138. (6) Tromp, R. H.; Jones, R. A. L. Macromolecules 1996, 29, 81098116. (7) Aymard, P.; Willams, M. A. K.; Clark, A. H.; Norton, I. T. Langmuir 2000, 16, 7383-7391. (8) Loren, N.; Hermansson, A.-M. Int. J. Biol. Macromol. 2000, 27, 249262. (9) Loren, N.; Hermansson, A.-M.; Williams, M. A. K.; Lundin, L.; Forster, T. J.; Hubbard, C. D.; Clark, A. H.; Norton, I. T.; Bergstrom, E. T.; Goodall, D. M. Macromolecules 2001, 34, 289-297. (10) Williams, M. A. K.; Fabri, D.; Hubbard, C. D.; Lundin, L.; Foster, T. J.; Clark, A. H.; Norton, I. T.; Loren, N.; Hermansson, A.-M. Langmuir 2001, 17, 3412-3418. (11) Amici, E.; Clark, A. H.; Normand, V.; Johnson, N. B. Biomacromolecules 2000, 1, 721-729. (12) Picullel, L.; Bergfeldt, K.; Nilsson, S. In Biopolymer Mixtures; Harding, S. E., Hill, S. E., Mitchell, J. R., Eds.; Nottingham University Press: Nottingham, 1995; pp 13-35. (13) Zhang, J.; Rochas, C. Carbohydr. Polym. 1990, 13, 257-271. (14) Morris, V. J. In Functional Properties of Food Macromolecules, 2nd ed.; Hill, S. E., Ledward, D. A., Mitchell, J. R., Eds.; Aspen Publishers Inc.: Gaithersburg, MD, 1998; pp 148-169. (15) Paoletti, S.; Smidsrod, O.; Grasdalen, H. Biopolymers 1984, 23, 1771-1779. (16) Chronakis, I. S.; Picullel, L.; Borgstrom, J. Carbohydr. Polym. 1996, 31, 215-225. (17) Hermansson, A.-M. Carbohydr. Polym. 1989, 10, 163-181. (18) Clark, A. H.; Eyre, S. C. E.; Ferdinando, D. P.; Lagarrigue, S. Macromolecules 1999, 32, 7897-790.
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