Influence of Nongelling Hydrocolloids on the Gelation of Agarose

Article pubs.acs.org/Biomac

Influence of Nongelling Hydrocolloids on the Gelation of Agarose Natalie Russ,* Birgitta I. Zielbauer, Kaloian Koynov, and Thomas A. Vilgis Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany

ABSTRACT: The combination of different gelling and nongelling hydrocolloids is known to yield complex systems with a wide range of mechanical properties. Here, the influence of the nongelling hydrocolloids sodium-alginate and xanthan on the gelation of agarose is investigated. The two polyelectrolytes differ significantly in their flexibility, leading to opposing effects on the thermomechanical properties of the resulting composite gels. The network structure of the agarose as well as viscoelasticity, gelling temperature, and thermal stability of the gels are altered. These properties are investigated by strain and temperature dependent oscillatory rheological measurements as well as confocal laser scanning microscopy. A phenomenological model to describe the network formation of agarose in the presence of alginate or xanthan respectively is presented.

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entropy is thus compensated by a gain of energy. Arnott et al.4 demonstrated by XRD and optical rotation measurements that the agarose gels are formed by double helical structures of the single polymer chains. The binding by hydrogen bond leads to a double helical combination of two single agarose chains.5 Further cooling causes aggregation of these double helices. Therefore, a two-step gelation mechanism for agarose has been proposed.6,7 First, the formation of a gel by joining the randomly distributed coils into a double helical association by hydrogen bonds, followed by the aggregation of the double helices into a tight three-dimensional network (Figure 3a). In between the junction zones of this network, meshes are established that enable the enclosure of water molecules by forming hydrogen bonds with the hydroxyl groups of the agarobiose units facing outward.4 The block copolymer alginate8,9 is a flexible polyelectrolyte with thickening ability in its dissociated state. This thickening effect results from an increasing dissociation of carboxyl groups and the simultaneously increasing electrostatic repulsions of the ionic groups along the polymer chain. The entangled coiled chains transform to a more elongated form and the internal friction of the solution increases. From a critical molar mass on, the polymer chains entangle and in the absence of external stresses, a permanent release and rebuilding of the entangle-

INTRODUCTION Hydrocolloids have a broad application range in different sectors, for example, in the pharmaceutical industry for encapsulation or as material of contact lenses. They are found in the cosmetic area as well to give some beauty products a certain texture and softness. Especially in the food industry their presence is ubiquitous as thickening additives, gelling agents, or stabilizers for dispersions and solutions. Different gelling agents are often used in combination with nongelling thickeners, to use synergetic effects and enhanced textural properties based on structural changes on nanoscales. The combination of a gelling agent with thickeners having significantly different physical properties concerning chain conformation, chain stiffness, and collective solution behavior can change the physicochemical properties of the gelling agent in different ways and enlarge its application range. In this study, the gelling and physical properties of agarose, as influenced by adding the nongelling agents alginate and xanthan, are investigated. Agarose forms a three-dimensional network while cooling an aqueous solution. Molecular and chemical properties of agarose are described elsewhere.1,2 In the liquid phase near the boiling point of water, the agarose molecules form a random coil conformation and are distributed homogenously in the solution.3 While gelling under cooling, this entropically preferred state has to be overcome and the single polysaccharide chains are forced to associate with other chains via hydrogen bonds. Below the gelling temperature the loss of © 2013 American Chemical Society

Received: August 27, 2013 Revised: October 15, 2013 Published: October 16, 2013 4116

dx.doi.org/10.1021/bm4012776 | Biomacromolecules 2013, 14, 4116−4124

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a defined geometry with a diameter of 25 mm and a height of 3 mm. Excess solution was wiped off with a broad razor blade. The solution was allowed to cool down to room temperature for 15 min, the molds were removed and the set gels hardened in the refrigerator at 4 °C for 24 h. To produce the mixtures of agarose−alginate and agarose− xanthan, the pure xanthan and respective alginate dispersions have been prepared first. For this, 1% w/w of the xanthan and alginate powder were dispersed in distilled water and stirred for 24 h at room temperature at 200 rpm. Then 1% w/w agarose was dispersed in distilled water like before and mixed in a 1:1 ratio with the hydrocolloid dispersions. The mixture was shaken by hand and heated up again to 90 °C for 5 min while stirring at 200 rpm. Afterward, the gels hardened like before first at room temperature and then in the refrigerator. Dynamic Viscoelastic Measurements. The viscoelastic properties as well as the sol−gel transition of the hydrocolloids were measured with a stress-controlled rheometer (Bohlin Gemini 200, Malvern, U.K.). Two different tests were performed: amplitude-sweep test and temperature-sweep test. For amplitude-sweep test an oscillating deformation was applied and the storage G′ and loss G″ modulus are measured in dependence of the applied strain γ in a range between 0.001 and 1 at a constant frequency f = 1 Hz and temperature T = 25 °C. As default parameters, an integration time of 5 s, a delay time of 0.5 s, and five periods were set. Measurements of five different samples were averaged and the standard deviation taken as error. The gelled agarose, respectively, mixture samples were investigated with plate−plate geometry. The upper stainless steel plate has a diameter of 25 mm. Here an adequate contact between samples and measuring system without significant compression of the sample has to be ensured. Therefore the gap size for each sample was set manually. After inserting the sample, the upper plate was set to gap of 3000 μm and lowered in steps of 100 μm until the contact between sample and plate was distinct and visible. To avoid slippage of the samples at high deformations, sandpaper with a grain size 80 was pasted onto both plates. Temperature-sweep test yields information about the sol−gel transition and the thermal stability of the hydrocolloids. Storage G′ and loss G″ modulus are measured as a function of temperature with constant frequency f = 1 Hz and strain γ = 0.001 or 0.01. The default parameters were the same as for the amplitude-sweep but an upper stainless steel plate with a diameter of 40 mm was used. The shown curves in Figure 4 are the averages of a 3-fold determination. A total of 1350 μL of the hot, liquid sample (see Sample Preparation without gel setting) were loaded to the preheated measuring system of 80 °C and the upper plate was lowered slowly to a gap of 1000 μm. The liquid sample was first cooled down from 80 to 20 °C and reheated to 95 °C at a rate of 1 K/min. Temperature control was enabled by a coaxial peltier cylinder system. This temperature control system consists of a cylinder system heated by peltier elements and a water cooling kit to allow the dissipation of the heat energy. Thus, a temperature stability of ±0.1 °C was achieved. To avoid water evaporation during the measurement, a thin film of paraffin oil (viscosity η = 25 mPas, Merck KGaA) protected the sample. To guarantee a continuous contact between sample and plates during gelling and melting, an autotension control was employed. This function allows maintaining a constant normal force on the sample during the solid phase such that expansion or contraction of the gels due to thermal variations can be compensated. Thus, a constant normal force of 0.1 N was applied on the sample and this should also compensate for the thermal expansion of the plates of 1 μm/°C. Confocal Laser Scanning Microscopy. Agarose was covalently labeled with 5-(4,6-dichlorotriazinyl)aminofluorescein (5-DTAF, CAS No. 51306−35−5) from invitrogen with maximum absorption at λab = 492 nm and maximum emission at λem = 517 nm. DTAF is often used to label polysaccharides because it is known to covalently bind to primary hydroxyl groups above pH 9 in aqueous solutions.15 However, in the literature, no description of its use to stain agarose has been found. Therefore, the following labeling procedure based on a procedure for labeling xylan with DTAF (http://www.fgsc.net/ fgn37/chalmers.html) was developed. A 1 g aliquot of agarose was

ments occurs until a constant density of entanglements is reached.10 In contrast, xanthan11,12 is a very stiff polysaccharide. Dissolved in cold water, xanthan forms highly viscous solutions with strong shear thinning behavior. The strong increase of the viscosity of a xanthan solution is described by Nordqvist and Vilgis6,13 by a simple model based on the rigid and ordered structure of xanthan. For simplification, xanthan is regarded as stiff and highly charged polysaccharide (in salt-free medium), which can be approximated as rod-like, charged molecule. The negatively charged side chains cause an electrostatic repulsion along the polymer chain whereby the molecules are forced to change into an elongated form and gain certain rigidity. Thus, the solution contains long, charged, stiff polymer chains with a certain effective excluded volume, defined by their contour length and thickness. At low xanthan concentration, the charged rods repel each other according to the Coulomb interactions, but are still able to diffuse. At larger concentrations, the competition between electrostatic repulsion and decreasing distance between the xanthan molecules creates frustrated conformations. At a critical concentration, this repulsion gets so strong that their motion is strongly hindered and they undergo a so-called jamming transition,14 the rods get immobilized at random positions with random orientations. This immobilization leads to a more solid-like behavior and, thus, a yield stress of the dispersion. Thus, the combination of the gelling agent agarose and the thickeners alginate, respectively, xanthan promises a multiplicity of possibilities due to their contrary physical properties, mainly the strong difference in chain stiffness and length scale. These will affect the molecular mesoscales, that is, textural properties which are determined on the size of the polysaccharides themselves. As a result, the physical properties of the resulting agarose-based gels are strongly altered. The aim of this study is to get insight into the mechanisms by which the presence of nongelling hydrocolloids modifies the gelation mechanism of agarose and influences the final thermomechanical properties of the resulting composite gels. To characterize those, amplitude and temperature-dependent oscillatory rheology measurements have been performed. Additionally, the network structure of the different gels was investigated by confocal laser scanning microscopy (CLSM), yielding additional direct structural information on a mesoscopic scale. To enable CLSM a new synthesis route to label agarose with a fluorescent dye was developed. Based on these results, simple phenomenological models are proposed to describe the gelation of agarose in combination with a hydrocolloid with flexible polymer chains (alginate) and a charged stiff and rod-like polymer (xanthan).

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MATERIALS AND METHODS

Materials. Agarose (CAS No. 9012−36−6) was purchased as fine white powder from Fisher Scientific GmbH with a specified gelling temperature (Tgel) between 34 and 45 °C. Na-alginate (CAS No. 9005−38−3) was purchased from Sigma-Aldrich Chemie GmbH. Xanthan gum (reinst, E-415) was from Carl Roth GmbH and Co. KG with the CAS-No. 11138−66−22 and reaches according to the producer in a 1% KCl solution a viscosity of 1200−1600 mPas. Sample Preparation. The 0.25, 0.5, 0.75, or 1% w/w of agarose was dispersed in distilled water. The dispersion was heated up to 90 °C and kept at this temperature for 5 min while stirring (200 rpm). By using a contact thermometer and a snap-cap vial, a constant temperature of 90 °C and minimal water evaporation could be assured. After 5 min, the hot clear solution was poured into molds with 4117


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Figure 1. Log−log plot of (a) elastic modulus G′ and (b) viscous modulus G″ vs strain at f = 1 Hz and T = 25 °C for 0.25, 0.5, 0.75, and 1% agarose, as well as for 1% agarose−alginate and 1% agarose−xanthan. suspended in 50 mL of distilled water and stirred at 95 °C until a clear solution evolved. The solution was cooled down to 50−60 °C, 0.008 g 5-DTAF, and 15 mL of a 1% w/v Na2SO4 solution were slowly added dropwise. The amount of fluorescent dye was estimated such as to label each 200th monomer to avoid a crucial influence on the gelation mechanism by the dye molecules. After the addition of 2−3 drops of a 10% w/v NaOH solution, the turbid yellow suspension cleared and an intensive yellow solution arose. This solution was stirred for over 2 h at 50 to 60 °C. Afterward 120 mL absolute ethanol was added and a fluorescent yellow precipitate could be observed. After cooling at −24 °C for 15 min the suspension was filtered and washed several times with cold ethanol resulting in a fine yellow powder. Samples for CLSM were prepared as described in Sample Preparation but substituting 20% w/w of the agarose by the fluorescently labeled one. A total of 500 μL of each hydrocolloid mixture were gelled inside eight-well chambered borosilicate coverglass systems (Nunc Lab-Tek, Thermo Fisher Scientific, U.S.A.). The network structure of the agarose gels and composite gels was analyzed using a commercial confocal laser scanning system FluoView 300 FV 300 (Olympus, Japan) in combination with an inverted microscope IX70 (Olympus, Japan). Fluorescence was excited by the 488 nm line of a 20 mW Argon laser fiber coupled to the laser scanning unit and collected after passing through a LP505 long pass emission filter. Images were taken at 30 μm sample depth with a 60× water immersion objective (UPLSAPO 60×W, NA 1.20, WD 0.28 mm, Olympus, Japan). The obtained CLSM images were analyzed using the “ImageJ” software package, including the “LOCI” plugin.

use of deformation energy to already deform subdomains of the gel structure before the inner structure finally breaks. Such predeformation can result, for example, from relative movements between molecules not embedded in the network or free chain ends. Thus, strictly spoken, the length of the linear viscoelastic range is limited by the increase of G″ and not by the decay of G′. However, due to the strongly dominating character of G′ for all investigated gels, we determined the linear range from G′, justified by the same tendency for G″ in Figure 1b. Pure agarose gels exhibit a consistent increase of the elastic modulus with increasing agarose concentration, as shown in Figure 2. The higher the agarose concentration, the more

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RESULTS AND DISCUSSION Viscoelastic Properties. The influence of the nongelling hydrocolloids with different chain flexibility on the viscoelastic properties of agarose was studied. During these measurements either the agarose or the total polymer concentration was kept constant. In the mixtures, the total polymer content was 1% w/ w. Figure 1 shows the elastic and the viscous modulus as a function of strain for the hydrogels composed of 0.25, 0.5, 0.75, and 1% w/w agarose, as well as the 1:1 mixtures of 1% w/w agarose−alginate and 1% w/w agarose−xanthan. G′ (Figure 1a) exhibits an obvious linear elastic range with a constant value of G′ for all systems and decreases as the applied strain gets too high. On the contrary, G″ (Figure 1b) shows a peak prior to the final decrease for all systems. This trend is typical for dispersions with networks or cross-linked gels.16 The increase of G″ prior to the drop of G″ indicates an increasing

Figure 2. Elastic modulus G′ vs agarose concentration at a constant strain γ = 0.01.

agarose helices can aggregate and the more junction zones and networks of higher elasticity are formed. It is nicely verified that at larger concentrations c > 0.5%, that is, agarose concentrations significantly larger than the critical gel concentration (c* = 0.2%1), the linear viscoelastic modulus increases linearly with the concentration, as suggested by the classical gel theory.17 The averaged elastic modulus for the different systems at a certain strain, γ = 0.01, as well as the strain at the end of the 4118


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Table 1. G′ and γmax for the Different Gel Systems G′ at strain γ = 0.01 (Pa) γmax

1% agarose

0.75% agarose

0.5% agarose

0.25% agarose

1% agarose−alginate

1% agarose−xanthan

8347 ± 603 0.059 ± 0.007

4949 ± 228 0.028 ± 0.004

1281 ± 91 0.057 ± 0.033

178 ± 9 0.090 ± 0.027

1616 ± 29 0.038 ± 0.013

883 ± 43 0.140 ± 0.063

Figure 3. Model of the gelation mechanism of (a) pure agarose, (b) agarose−xanthan mixture, and (c) agarose−alginate mixture: fine black helices and coils, agarose; gray cylinders, xanthan rods; thick gray coils, alginate polymer chains. In the agarose−xanthan mixture the stiff and rigid xanthan rods disturb the coil and helix diffusion of agarose resulting in a less elastic network. In the agarose−alginate mixture, the interpenetrating alginate coils are embedded within the agarose network and act as fillers by stabilizing and increasing the elasticity of the agarose gel.

do not show a significant temperature dependence,6 which indicates that the time scales of motion of the xanthan molecules is very slow. The jammed structure of the xanthan rods hinders the free diffusion of the agarose chains, and the formation of helices needs to be considered in a restricted (phase) space that is formed by the random array of stiff xanthan molecules. Thus, it is assumed that during the gelation less agarose coils can transform into helices and, hence, less helices can agglomerate, and the resulting gel structure consists of less and weaker junction zones (Figure 3b). Consequently, the set gels have a lower elastic modulus. This could be understood in a naı̈ve “scaling” ansatz. The simplest expression for the modulus of a cross-linked and purely entropy elastic gel compares the thermal energy kBT with the typical mesh size, ξ, via a dimensional analysis G0 ∝ kBT/ξ3. Rods with their length, L > ξ, form at random jamming, an array of rods with a typical density of the order of L−3. Comparing these length scales, a larger mean mesh size is of the order of ξ + L (apart from a prefactor), and the modulus of the mixed systems is likely to become lower compared to the pure agarose network. However, as it will be shown below by confocal microscopy, the mesh size seems to become smaller, which shows a complex gel formation in the mixed agarose−xanthan system. As explained above, the xanthan immobilization starts already at high temperatures, above the gelling temperature of agarose. Upon further cooling, the agarose network needs to form itself in a restricted space, spanned by the randomly oriented stiff

linear viscoelastic (LVE) range (γmax), are given in Table 1. The latter was defined as the strain at which G′ had dropped to 90% of its value at γ = 0.01. As can be seen in Figure 1b, the length of the plateau value for G″ follows the same trend as discussed below. Regarding the composite gels, distinguished differences in elasticity and deformation strength for alginate and xanthan addition can be observed. Adding the nongelling polymer alginate to a 0.5% agarose solution up to a total polymer concentration of 1% slightly increases the elastic modulus, while the addition of the same amount of xanthan leads to a decrease in the modulus and also significantly increases the width of the LVE range. The lower elastic modulus of the agarose−xanthan gels in comparison to the pure agarose gels results from the molecular arrangement in the xanthan dispersions. As already described, the rigid xanthan molecules undergo a jamming transition due to electrostatic repulsion6 under the preparation conditions of the mixtures. These have been prepared by mixing the already set xanthan dispersion with the hot agarose solution. The presence of the stiff xanthan chains during network formation implies a strong limitation of diffusion and mobility for the agarose chains. While heating the mixture, the agarose has to be dissolved in the array of the xanthan chains. The dynamics of the jammed xanthan structure is much slower compared to the motion of the flexible agarose chains. In addition, it is not much affected by heating, because the viscosity of xanthan solutions 4119


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Figure 4. Oscillatory measurements of G′ and G″ during cooling and reheating ramps as well as tan δ vs temperature at f = 1 Hz and constant strain γ for (a, b) agarose, (c, d) agarose−alginate, and (e, f) agarose−xanthan. In (a) the measurement curve of agarose in 0.5% concentration (gray curves) is shown as well. Additionally, the evaluation of the gelling point is demonstrated for all systems (b, d, f).

xanthan molecules. As the mesh size ξ of the pure agarose network is smaller than the length L of the xanthan rods, strong heterogeneities in the agarose network structure result. Helical cross-links may form a compact network inside the xanthan “meshes” of volume L3 but also subsequent cross-links connecting at scales Ξ larger than the typical xanthan distances Ξ > L. At small amplitudes and low frequencies, the network elasticity is dominated by the network of larger meshes Ξ. For both types of cross-links, in and outside the typical volume L3, the aggregation of helices is disturbed compared to pure agarose gels. The low deformation modulus of the wider network formed at scales Ξ should then be lower than that of a pure agarose gel, as observed by the experiments in Figure 1. As L > ξ, Ξ is larger than ξ and can be crudely estimated as Ξ ≈ ξ(1 + ξ/L), which satisfies the requirement that for large L the

mean distance between the rods becomes larger (due to strong electrostatic repulsions) and Ξ approaches ξ again. The modulus G ∝ kBT/Ξ3 can then be estimated as G ∝ kBT/ξ3 (1 − 3ξ/L), which is slightly lower than the original modulus for the pure 0.5% agarose network. Note that this process of restricted (nonequilibrium) network formation does not correspond to the usual reinforcement of elastomers, which will lead usually to an enhancement of the modulus.18 In contrast, the mixture with alginate shows higher values of the elastic modulus than 0.5% agarose gels. Alginate in solution has a structure of flexible, mobile and entangled coils of polymer chains which hardly affect the agarose helix diffusion. During gelation the aggregation of agarose double helices and the build-up of the three-dimensional network are hardly 4120


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Figure 5. CLSM images of agarose gels with (a) 1, (b) 0.75, (c) 0.5, and (d) 0.25% concentration.

hindered.19 The entangled flexible polymer chains of the alginate are easily incorporated into the agarose meshes and act similar as soft filler particles in the network (Figure 3c). Thus, the agarose network formation is less restricted compared to the formation when xanthan is added. Such swollen networks were already observed for other mixtures of a gelling agent with nongelling hydrocolloids.20 Effectively, the flexible alginate molecules provide larger entropy, which yields a larger modulus. In addition the embedded flexible alginate chains act as reinforcing “filler” self-avoiding walk shaped particles in the network.21 The composite agarose−xanthan shows the longest LVErange of all studied gels in Figure 1a, as well as in Figure 1b. Regarding the error margins of γmax of the pure agarose gels and the agarose−alginate mixture, no significant differences can be distinguished. This means that the mixture agarose−xanthan can resist a stronger deformation as the pure agarose gel or the mixture agarose−alginate, which is in accordance with the model depicted in Figure 3. The jammed xanthan rods arrange around the agarose network and “freeze” at their ordered transition. To deform the agarose network, first the yield stress of the dispersion of blocked xanthan rods has to be overcome. Second, the agarose network is less strong, because the helix aggregates are less pronounced and induce a more flexible and deformable network. In conclusion, the mixture with alginate increases the elasticity of the agarose gels compared to the 0.5% agarose gel, while the mixture with xanthan improves their stability against deformation.

Sol−Gel Transition. The following section discusses the sol−gel transition and thermal stability of the different composite gels. These were probed by temperature-dependent oscillation measurements in the linear viscoelastic region. For this, the hot liquid samples were placed on preheated plates, subsequently cooled, and finally reheated in the rheometer. Figure 4a, c, and e show the elastic and viscous moduli during this temperature cycle for pure agarose, agarose−alginate, and agarose−xanthan gels. In the initial hot, molten state at 80 °C, agarose behaves as a low viscosity liquid with G″ > G′ and tan δ > 1. During cooling, the agarose molecules assemble into helices and finally aggregates of helices, leading to a steep increase in G′ and G″, and resulting in a stable gel with solid character,22 where G′ > G″ and tan δ < 1. As expected for agarose,7,23 a large hysteresis during reheating is observable, resulting from the fact that the aggregates require a large amount of energy to be molten up. Frequency dependent measurements of G′ and G″ would be the most accurate method to determine the gel point; however, they are timeconsuming and difficult to perform in the vicinity of the gel point due to the time-dependent nature of the gelation process. As proposed by Winter et al.,24 as well as by Labropoulos et al.,25 the gelling point Tgel, where the sol−gel transition occurs, can be determined as the temperature where tan δ = G″/G′ = 1 and an abrupt rise in G′ and G″ can be observed.16 However, due to the low viscosity of agarose in the sol state, the sensitivity of the rheometer was not sufficient to accurately determine G′ in the sol state, and the crossover of G′ and G″ is masked. G′ is overestimated for this case (Figure 4a, cooling cycle T > ∼36 °C) due to inertia effects. Therefore, it has been 4121


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chosen to determine Tgel by extrapolating tan δ to 1, as shown in Figure 4b. However, it has to be mentioned, that the uncertainty of this method is relatively high due to the far extrapolation needed. Although the crossover was directly detectable for the case of the agarose−alginate mixture due to its higher viscosity, it has been chosen to evaluate Tgel in the same way as for the pure agarose system by taking the temperature where tan δ = 1 (Figure 4d). On the contrary, for the system agarose−xanthan, the system exhibits elastic character even in the sol state, because xanthan is at the applied low amplitude a weak gel itself and dominates the modulus in the mixed system in the sol state with G′ > G″. Therefore, no crossover of G′ and G″ is expected, and the exact gelling point of agarose is masked. However, tan δ, which shows a slow decrease during cooling, starts dropping at a much faster rate at a temperature, where G′ and G″ show a steep increase. This temperature is therefore assumed to indicate the gelation of the agarose network and was taken as Tgel for the system agarose−xanthan (Figure 4f). As a result, the mixed gels show smaller values for Tgel than the pure agarose. As the gelling temperature of agarose is concentration-dependent, having lower values for lower concentrations,3,7 the gel setting occurs at lower temperatures for the mixed gels, which only contain a concentration of 0.5% agarose as the only gelling agent. The lower number of agarose helices and aggregates in the solution requires more time for the final network to be formed. No significant differences in Tgel for added alginate and xanthan are found. The gelling point of the agarose-xanthan mixtures is slightly lower compared to the one for the mixture with alginate, but due to the different determination methods, a comparison is difficult. From the reheating curves in Figure 4, differences in the thermal stability of the pure agarose gel and the composite gels can be observed. The function G′(T) of the agarose−alginate gel shows a strong decline already at T = 62 °C, whereas the 1% agarose gel is more heat stable. Because a pure agarose gel of 0.5% shows similar heat stability than the one of 1% (Figure 4a), the decrease in heat stability for the agarose−alginate gel cannot be attributed to the lower concentration of agarose present in the mixture. It may be a sign of a slight weakening of the agarose network junctions due to the presence of alginate, but less than in the case of the rigid and ordered structure of xanthan. The small hindrance of the alginate chains in the mixture gets only visible at high energy. However, due to the presence of the flexible alginate chains inside the network meshes, this does not lead to a weakening of the gel structure at temperatures below Tgel. On the contrary, the agarose−xanthan mixture shows an even lower thermal stability than the mixture with alginate. The elastic modulus starts to decay already at T = 42 °C. In comparison to the mixtures with alginate, the mixture with xanthan strongly hinders the diffusion and helix formation of agarose molecules and leads to less elastic and stable gels (see Viscoelastic Properties) with a corresponding low thermal stability. Thus, these composite gels already begin to melt at lower temperatures. Confocal Laser Scanning Microscopy. CLSM images were taken to investigate the network structures of the agarose gels and the mixed gels containing alginate and xanthan as well as the distribution of the thickeners in the agarose network. For this purpose, agarose was labeled with the fluorescent dye 5DTAF having an emission maximum of λem = 517 nm. Alginate, xanthan, or water do not exhibit emission lines in the detected

wavelength range; thus, the green highlighted regions in the CLSM images result only from agarose. Figure 5 shows the CLSM images of the pure agarose gel at four different concentrations. In all images the formation of a network gets clearly visible. The meshes of the network can be identified by the dark spots, surrounded by highlighted structures. These dark spots are generated by the water embedded in the network meshes. Pernodet et al.26 examined agarose gels of different concentrations by AFM. With increasing agarose concentration these authors found a decreasing pore size of the network. The averaged pore size of a 2% agarose gel was 364 nm and of a 5% gel 201 nm. Narayanan et al.27 determined agarose gel pore size via absorbance measurements and found a decreasing pore size with increasing agarose concentration as well. They suggested a power law dependence between pore size and concentration ξ ∝ cν with an exponent ν ≈ 1.6. Figure 5 suggests as well a decrease in pore size with increasing agarose concentration. By comparing the results of the dynamic viscoelastic measurements with the change of pore size, it gets obvious that the elastic modulus G′ correlates with the inverse pore size ξ. With increasing agarose concentration the elastic modulus of the gel increases, while the pore size decreases. According to the simplest theory for cross-linked purely elastic gels, the modulus is related to the pore size via G0 ∝ kBT/ ξ3. To test if this relation holds for the agarose gels investigated here, the pore sizes for different concentrations where estimated from the CLSM images by manually measuring the roughly dark round structures assumed to represent the pores of the network. A total of 150 points were measured per concentration. The averages are shown in Table 2. Table 2. Average Pore Sizes ξ and ξ−3 for the Different Concentrated Agarose Gelsa

pore size ξ (μm) ξ−3 (μm−3) a

0.25% agarose

0.5% agarose

0.75% agarose

1% agarose

1.55 ± 0.36

0.99 ± 0.21

0.73 ± 0.18

0.56 ± 0.13

0.27 ± 0.19

1.03 ± 0.65

2.61 ± 1.94

5.69 ± 3.89

Standard deviation is given as errors.

To compare the resulting concentration dependence when assuming G0 ∝ 1/ξ3 with the concentration dependence of the experimentally obtained elastic modulus this quantity was computed and both results were normalized by their value for the 0.25% gel. The results are given in Table 2 as well. Figure 6 shows normalization of the modulus estimated from the pore sizes ξ−3/ξ0.25%−3, as well as the normalized elastic modulus G′/G′0.25% in dependence on the agarose concentration. Both curves show an approximately linear increase for c > 0.5% with increasing temperature, indicating a qualitative validity of the model for elastic gels in this case. Figure 7 shows the comparison of the 1% mixtures agarose− alginate 1:1 and agarose−xanthan 1:1. The addition of the thickeners increases the homogeneity compared to the 0.5% agarose gel (Figure 5c) enormously. Also, differences between alginate and xanthan can be recognized. In the mixture with alginate, the formation of the agarose network seems to be more pronounced. Comparing to the 0.5% gel, the addition of the thickener stabilizes the weak network of the agarose gel and the network meshes are more homogeneous and equally distributed. The entangled and flexible alginate polymer chains 4122


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the contrary, addition of the rod-like stiff molecules of xanthan on the one hand hinders the diffusion and helix formation of the agarose chains, leading to a gel with lower elasticity but higher resistance against strain. For the first time, confocal microscopy allows imaging the network structure of the agarose gels and the hydrocolloid composites. This allows the determination of the pore size in dependence on the agarose concentration. In general, this study demonstrates that hydrogels consisting of an agarose network can be modified by adding thickeners like alginate and xanthan. Thus, material specific properties like viscoelasticity, gelling temperatures, and thermal stability can be adapted and the scope of application enlarged.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

Figure 6. Converted and normalized pore size and normalized elastic modulus vs agarose concentration.

Notes

The authors declare no competing financial interest.

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are embedded in and between the network meshes of agarose and the network formation is assured. Xanthan in contrast forms rigid obstacles and restricts the complete gelation of the agarose. The simple model for the correlation between pore size and elastic modulus cannot be applied here anymore. As already described in the intersection of the Viscoelastic Properties, the gel composition of the agarose−xanthan mixture is more complicated due the different sizes of network structures and cannot be derived from the micrographs.

ACKNOWLEDGMENTS The authors would like to thank Andreas Best for fruitful discussion concerning the evaluation of the CLSM pictures and Andreas Hanewald for the technical support with the rheometer. The authors would like to thank as well Dimitri Merger (Institute for Chemical Technology and Polymer Chemistry) for the supporting and helpful measurements at the KIT. Special thanks to Sania Maurer and Gustav Waschatko for helpful discussions and support.

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CONCLUSION This study examines the influence of the presence of nongelling hydrocolloids with different physicochemical properties such as alginate and xanthan on the gelation behavior of agarose and the mechanical properties of the resulting gels. Amplitude and temperature dependent rheological measurements yield information about the interactions between agarose and alginate respectively xanthan. It is shown that, at equal polymer concentrations, the effect of the addition of a nongelling hydrocolloid to agarose depends strongly on the flexibility of the individual hydrocolloid chains. Adding flexible alginate chains enhances the elasticity of the agarose gels by embedding flexible alginate chains into the meshes of the agarose network, where they act similar to reinforcing “soft filler particles”. On

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Figure 7. CLSM images of (a) 1% agarose−alginate 1:1, (b) 1% agarose−xanthan 1:1. 4123


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Influence of Nongelling Hydrocolloids on the Gelation of Agarose

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