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water interface
Colloids and Surfaces A: Physicochem. Eng. Aspects 331 (2008) 48–55
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Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Influence of concentration and ionic strength on the adsorption kinetics of gelatin at the air/water interface S. Domenek a,∗ , E. Petit a , F. Ducept a , S. Mezdour a , N. Brambati b , C. Ridoux b , S. Guedj b , C. Michon a a b
UMR Ingénierie Procédé Aliment, AgroParisTech-INRA-CNAM-CEMAGREF, 1 avenue des Olympiades, 91744 Massy Cedex, France Rousselot, Chemin Moulin Premier, 84808 Isle sur la Sorgue Cedex, France
a r t i c l e
i n f o
Article history: Received 31 January 2008 Received in revised form 11 June 2008 Accepted 13 June 2008 Available online 22 June 2008 Keywords: Gelatin Intrinsic viscosity Adsorption kinetics Air/water interface Foam
a b s t r a c t The adsorption kinetics of gelatin to the air/water interface is still not fully understood. We investigated two samples of gelatin having different gel strength (65 Bloom and 280 Bloom), named, respectively gelatin 65B and 280B. The gelatin samples in solution were characterised by viscosimetric methods. We evidence a chain expansion of gelatin upon dilution due to the polyelectrolyte effect. The measurement of the intrinsic viscosity in water gave values of 22 mL/g for the sample 65B and 70 mL/g for the sample 280B. Although gelatin is a highly polymolecular polymer containing branched chains, the normalisation of the concentration by the intrinsic viscosity allowed the construction of a master curve of viscosity, giving the reduced critical overlap concentrations c* ∼ 0.6 and c** ∼ 5. All concentrations of the gelatin solutions used for the determination of the adsorption behaviour were within the range of the dilute regime. The analysis of the adsorption behaviour leads us to the hypothesis that there are two different phenomena: first, a step governed by diffusion to the interface which lasts until full coverage of the interfacial film. The length of this induction period seemed to be dependent on the gelatin chain length, with larger chains taking more time. Second, the adsorption of the chains to the air/water interface leads to the formation of an encumbered subsurface layer which opposes steric hindrance to newly arriving chains. This causes a continuous slow down of the rate of surface pressure increase, which was linear on the logarithmic time scale and showed a slope of −1. The adsorption kinetics was independent of the salt concentration of the solvent and of the gelatin chain size measured by the intrinsic viscosity. The gelatin sample 65B shows furthermore higher equilibrium surface pressures at high bulk concentrations (>0.01 wt%) than sample 280B, while the contrary was observed at small concentrations. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Gelatin is widely used in food industry for the stabilisation of foamed products because of its surface-active and gelling upon cooling. For the formulation of foamed products it is important to understand the behaviour of gelatin at the air/water interface. Foaming of gelatin solutions is often done at thermal conditions higher than the gelling temperature. In this case differences between gelatin samples can be observed. In order to discriminate the effects of the surface activity of gelatin from its gelling properties, it is necessary to study its interfacial behaviour in nongelling conditions. The adsorption onto the air/water interface of heteropolymers containing hydrophobic segments, such as pro-
∗ Corresponding author. Tel.: +33 1 69 93 50 68; fax: +33 1 69 93 50 05. E-mail address: [email protected] (S. Domenek). 0927-7757/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2008.06.034
teins, is a very much slower process than the adsorption of small surfactants owing to the much greater size and structural complexity. In the case of macromolecules, it may take several hours to reach the equilibrium. As a consequence, the time dependent interfacial properties play an important role in industrial processes. Gelatin, the hydrolysis product of structural collagen, has a very particular structure among proteins. It presents a high structural complexity because it has a large size distribution of the protein chains which are mainly linear although there is a small percentage non-hydrolyzed bonds giving rise to branched chain. However, it has no well-defined spatial conformation like globular proteins. It can thus also be considered as an ampholytic polymer with an isoelectric point varying between pH 7.5 and 9.3 for type A gelatin and 4.7 and 5.2 for type B gelatin. At temperatures higher than 40 ◦ C it has a loosely packed coil structure. Upon cooling at less than 40 ◦ C, the gelatin chains can undergo a conformation change and arrange themselves in triple helices, which might change the foaming behaviour of gelatin.
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Generally, the adsorption behaviour of proteins on the interfaces is described as a diffusive process with an energy barrier, including a phase of attachment of the proteins on a cleared interface area and a phase of rearrangement and denaturation in the adsorbed layer [1]. Sato and Ueberreiter [2] studied the adsorption of gelatin at the air/water interface at 40 ◦ C for different concentrations. They conclude that the surface concentration of gelatin is approximately 14-fold higher than the concentration in the bulk solution. Depending on the concentration of gelatin, the authors define different degrees of intermolecular interactions of gelatin molecules on the surface. At low surface concentrations there seemed to be a higher structuring of the gel than at higher concentrations (above 0.25 mg/mL). Several studies have been published on the behaviour of gelatin at the air/water interface, where the solutions were kept at room temperature. In this case, the gelatin molecules are able to form triple helices. The rate constants of the adsorption kinetics were calculated with the help of a first order exponential law analogous to the Avrami equation, which has the inconvenience that the equilibrium surface tension intervenes strongly in the calculation, which is generally difficult to determine experimentally. The relationship between foam volume and adsorption kinetics of various proteins was studied at room temperature [3]. A single, linear relationship between foam volume and rate constants was observed for different globular proteins. Gelatin, however, showed a distinct behaviour, having, relatively to the other proteins, a slow adsorption rate, but a high foam volume. This behaviour was attributed to its molecular properties and the solution viscosity being different from globular proteins [3]. The formation of a gel structure at the interface by gelatin has been evidenced by the measurement of the shear viscosity of gelatin/␣-casein mixtures at the interface [4], and by investigation of thin gelatin films with the help of atomic force microscopy [5]. The structural parameters playing obviously a role in the surface activity, it has been shown for surface active polymers such as poly(ethylene oxide) that the equilibrium surface concentration of the polymer, expressed in mol/m2 , diminished with an increase of molecular weight. However, for a polymer chain length higher than 2 × 103 the surface concentration became independent of the bulk concentration [6]. Lin et al. [7] investigated the interfacial dynamics of gelatin solutions together with different surfactants at 25 ◦ C when gelatin is in helix conformation. They showed that there was a long relaxation time leading to a gradual increase of the interfacial tension which was attributed to intraand interchain rearrangements of the gelatin chains occurring at the interface. Although there are data and descriptions of kinetics of the gelatin adsorption behaviour published, only Lin et al. [7] proposed a possible mechanism to understand the adsorption behaviour. For their analysis they considered gelatin being a linear polymer with a molar weight between 1 and 2 × 105 . It is commonly admitted that the first step of the polymer attachment on the interface is diffusion controlled. They showed, furthermore, that gelatin had a long relaxation time signature, which they attributed to the intra- and interchain rearrangements of the molecules at the interface may be a process analogous to the gel formation in bulk solution. Foaming is a dynamic process. Therefore, it is interesting to investigate the adsorption kinetics in further detail. The understanding of the kinetics can help to get more insight into the physical mechanisms of the adsorption process. Because industrial foaming processes are often carried out at temperatures higher than the conformation change (at 40 ◦ C) leading to the gel formation by gelatin, we worked here at temperatures higher than 40 ◦ C, in order to keep gelatin in its coil conformation. We used two different gelatin samples, distinguished by their gel strength. The viscosity of the gelatin solutions was investigated in order to determine the limits of the different concentration regimes. The
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adsorption kinetics of both samples was subsequently measured at different concentrations in the dilute regime. Two solvents were used differentiated by their salt content, but not by their pH in order to investigate the polyelectrolyte behaviour of gelatin samples. The kinetics of the recorded surface tension was analyzed. 2. Materials and methods All chemicals were purchased from Sigma–Aldrich (France). The gelatin samples (type A, acid hydrolysis) were provided by Rousselot SA (France). Sample 65B and 280B have gel strengths of 65 and 280 Bloom, respectively. The pH of both gelatin samples after dissolution in water was between 4.8 and 5, their isoelectric point being at approximately pH 8.5. Solutions were prepared in dionised water or in an acetate buffer (pH 5, 0.2 mol/L) in order to control the ionic strength of the solution. The mineral contents of sample 65B and 280B were 16.7 and 5.8 mg/g, respectively corresponding to, in 1 wt% gelatin solutions prepared in deionised water, at most about 2 × 10−5 and 0.7 × 10−5 mol/L of free ions. These extremely low ion concentrations correspond to less than 0.03% of the ionic strength of the buffer solution which can be neglected. Moreover, they can be also reasonably neglected in deionised water solutions when studying concentrations below 1 wt%, in the diluted regime. In order to melt the triple-helix aggregates of gelatin, the solutions were heated at 50 ◦ C for 30 min prior to the measurements. All solutions were prepared freshly each 3 days and stored at 4 ◦ C. The determination of the viscosity of the gelatin samples in the low concentration range at 44 ◦ C was carried out using a capillary viscosimeter equipped with an automatic dilution system (Viscologic, Laser Diffusion Systems, France). The determination of each time of passage through the capillary was done in 10 replicates and each solution was at least duplicated. The determination of the viscosity of the gelatin solutions at higher concentrations (≥0.1 wt%) was realised using a Rheometrics Fluid Rheometer (RFS II, TA Instrument) working with imposed deformation. The instrument was fitted with coaxial cylinders (R1/R2 = 0.96). A flow curve was carried out in the shear rate range 0.1–150 s−1 . The viscosity value was taken in the Newtonian plateau. The reduced viscosity (red ) of the samples was calculated after red =
− solvent 1 solvent C
(1)
where is the measured viscosity, solvent the viscosity of the solvent (water or acetate buffer), and C is the concentration (g/100 mL). Each concentration was measured in triplicate. The density of the diluted solutions was equal to water. Therefore we considered the weight percentage in the dilute regime equal to the unit g/100 mL. For concentrations higher than 1 g/100 mL we worked in wt%. The measurement of the surface tension of the gelatin solutions was carried out with an automated tensiometer (Tracker, IT Concept, France). The principle of the measurement is based on axisymmetric drop shape analysis of an air bubble at the end of a capillary in the solution containing the surfactant. The surface tension is calculated with the help of the Laplace equation. The bubble volume was 9 L and the temperature of the gelatin solution was maintained at 42 ◦ C with the help of a sample holding device equipped with double wall jacket and branched to a water bath. The temperature of the solution and the surface tension were recorded online. In order to avoid the evaporation of the solution a layer of paraffin oil was placed on the surface of the gelatin solution. The non-miscibility of water and paraffin oil and the non-perturbation of the measurement were verified during several hours with deionised water. The surface tension of water and the acetate buffer were checked each day. In the following the decrease of the surface tension is expressed as an increase of surface pressure
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Fig. 1. Reproducibility of the adsorption kinetics for four repeat experiments of gelatin 65B at 0.1 and 0.001 wt% in acetate buffer.
( = solvent − gelatin ) in order to normalise small variations in the solvent surface tension. Fig. 1 shows the reproducibility of the evolution of the surface pressure versus the logarithmic time scale for one gelatin sample (65B) at two concentrations. We observe that the experimental error on the data is larger at short times, where we have an uncertainty of 2 mN/m, and the curves at zero surface pressure are noisy. However, the overall shape of curves is reproducible and the end values show smaller error. Due to the long experiment time required to approach equilibrium values (>10 h) and the sensibility of the experiment to external factors, the equilibrium value was not approached for each experiment. For analysis, the following parameters were quantified: (i) the initial surface pressure (˘ i ) read at time zero, the length of the induction phase (tlag ) quantified by the calculation of the crossing point between the initial straight regression line and the tangent to the surface pressure increase, and the value of the equilibrium pressure (˘ eq ) calculated with the help of a sigmoid line fit (logistic function with four parameters), in the case that the beginning of the final plateau was observed. The quality of the fit was tested on the surface tension curves, where a final plateau value was observed. For differentiation, curves were interpolated on the logarithmic time scale with 90 points and a moving average was calculated over 10 points. The derivative was filtered by suppressing negative and zero values. Computing was done with Matlab v6 software (Matworks Inc.).
Fig. 2. Evolution of the reduced viscosity (red ) of the gelatin solutions versus concentration at two salt concentrations. Symbols: gelatin 65B in acetate buffer (), gelatin 65B in water (䊉), gelatin 280B in acetate buffer (), and gelatin 280B in water ().
determination yields an apparent average value being an average of the gelatin chains contained in the sample. Fig. 2 shows the evolution of the reduced viscosity with the concentration in both solvents, water and acetate buffer at the same pH for the two gelatin samples. The 280B sample has a higher reduced viscosity than the 65B sample, meaning that it contains in average larger molecules, which is consistent with its higher Bloom value. Both samples show an increase of the reduced viscosity upon dilution in water, which is a typical behaviour of polyelectrolytes. The increase of the ionic strength of the solvent (acetate buffer 0.2 mol/L) without change of the pH of the gelatin solutions decreases successfully the expansion confirming the polyelectrolyte effect hypothesis. However, even in the acetate buffer solvent, the small remaining expansion of gelatine chains in diluted conditions prevents us from extrapolating the reduced viscosity to zero gelatin concentration using the Huggins equation. The intrinsic viscosity was, thus, calculated using the relationship developed by Kravtchenko and Pilnik [8] for polyelectrolytes, which allows the extrapolation to [] from the plateau of the reduced viscosity at higher concentrations. The intrinsic viscosity was computed from the gelatin concentration (C) of 0.5 wt% using Eq. (2): [] =
3. Results and discussion 3.1. Determination of the intrinsic viscosity and the critical overlap concentration In order to characterise the gelatin samples, we studied the viscosity of the bulk solution. The ability to increase solution viscosity at a given concentration of polymers is dependent on the macromolecular size, rigidity and degree of branching, which contribute both to its hydrodynamic volume. Gelatin develops high solution viscosities compared to other proteins due to its flexibility and, thus, to its expanded coil structure. A method to determine the hydrodynamic volume of a polymer chain is the measurement of its intrinsic viscosity ([]), i.e. its hydrodynamic volume in diluted conditions. Because gelatin is a highly polymolecular polymer, the
1 2 C
solvent
− 1 − ln
12
(2)
solvent
The results are given in Table 1. The values of [] are much higher than those observed for globular proteins, such as soybean protein (0.05 dL/g [9]), but considerably lower than the intrinsic viscosity of polysaccharides used for stabilisation purposes in the food industry, such as locust bean gum (15 dL/g [10]) or xanthan (49.3 dL/g [11]). A marked difference in the average hydrodynamic volume is observed Table 1 Apparent intrinsic viscosity [] of the gelatin samples calculated after Kravtchenko and Pilnik [8]
Gelatin 65B Gelatin 280B
[] (dL/g) Acetate buffer
[] (dL/g) Water
0.22 ± 0.01 0.50 ± 0.01
0.27 ± 0.01 0.70 ± 0.07
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the polyelectrolyte effect, which increases the viscosity at small concentrations due to the chain expansion. Fig. 3b shows that the reduction of the concentration axes by the apparent intrinsic viscosity value yields a good adjustment of both samples curves to a single master curve, although gelatin chains have a broad size distribution. The transition zone between diluted and concentrated dilution regime stretches from 0.6 to 5 on the reduced concentration scale (Fig. 3b). The slopes are lower than the slopes observed for other biopolymers in coil conformation. For example, guar gum yields a slope of 1.2 in the dilute regime, 2.4 in the semi-dilute, and 5.4 in the concentrated regime [12], and the more rigid xanthan gum 1.25, 2.1, and 4.2, respectively [11]. Gelatin, in comparison to polysaccharides, is composed of more flexible chains. This flexibility brings about a higher capacity of expansion or contraction of the chains in function of their concentration in solution. In order to investigate whether the chain expansion of gelatin had an influence on the adsorption mechanism on the interface and in order to be able to detect the initial evolution of the surface pressure when gelatine starts to adsorb, we choose to work with both samples in the dilute concentration regime. 3.2. Development of the surface pressure in function of the gelatin concentration and the ionic strength
Fig. 3. Relationship between reduced viscosity and gelatin concentration. Reduced viscosity versus bulk concentration (a), reduced viscosity versus the bulk concentration scale reduced with the intrinsic viscosity calculated after Kravtchenko and Pilnik [8] (b). Symbols: gelatin 65B (䊉) and 280B () dissolved in water.
between both gelatin samples. The volume decrease of gelatin 280B chains in acetate buffer compared to water is much higher than the one of gelatin 65B. Generally, the larger the chain length of a polymer, the higher is its expansion ability. In order to specify the limits of the dilute regime of the gelatin solutions, the reduced viscosity was measured in a larger concentration range for both samples dissolved in water. This treatment allowed choosing the concentration range for the following tensiometric measurements. The results are shown in Fig. 3a. In concordance with [] difference, the change from the dilute to the concentrated regime is shifted to higher concentrations for sample 65B. A critical overlap concentration of approximately 3 wt% for gelatin 65B and 1.5 wt% for gelatin 280B is read (Fig. 3a). The slopes of the reduced viscosity–concentration curve in the dilute regime are smaller than slopes generally found for other polymers. Andrade et al. [10] measured 1.2 for locust bean gum in the dilute regime. The measured slope is most likely underestimated due to
The rate of surface occupation and the ability to develop a high surface pressure of a surface-active macromolecule are of importance in the foaming process. In order to study the phenomenon and to deepen our understanding of the adsorption process, the kinetics of the adsorption of gelatin to the air/water interface was investigated in a concentration range from 1 × 10−5 to 1 wt%. In Fig. 4 the increase of the surface pressure of gelatin 65B in acetate buffer and water is plotted on the logarithmic time scale as an example. All curves show a sigmoid shape with an induction phase. It follows a steep increase in surface pressure and a final phase approaching the thermodynamic equilibrium between the adsorbed layer and the bulk solution. The adsorption behaviour of globular proteins shows generally a comparable shape, although the induction phase is rarely observed for proteins. This is mainly due to the utilisation of high protein concentrations. For smaller concentrations, lag-phases can be observed [13]. Generally, the adsorption kinetics is described by an exponential decay [1,3]. The interpretation of the curve shape proposed is an induction phase due to diffusion to the interface, an adsorption phase of the proteins on the interface involving penetration and anchoring on a cleared interface area, and a phase of slow rearrangement of the molecules at the interface related to reorientation and conformational change [1]. In many adsorption studies on proteins, the rates of adsorption have been shown to be lower than the rate of diffusion [1]. In order to account for this behaviour, it was proposed that there is an activation energy existing at the interface for adsorption of the solute from the subsurface [14]. This energy barrier might be related to the denaturation of the proteins at the surface. Gelatin however, being already a denatured product of collagen, has no defined special conformation such as globular proteins. Moreover, in our experiments the gelatin solutions were kept at 42 ◦ C which keeps the molecules in their coil state. Therefore, gelatin could be considered as a linear polymer including a small number of branched chains. The form of the adsorption curve plotted in Fig. 4 is classically observed for the adsorption of polymer surfactants to interfaces, such as poly(ethylene oxide) [6], copolymers of poly(propylen glycol) [15], grafted poly(acrylic acid) [16], or biopolymers such as hydroxypropyl cellulose [17]. Furthermore, comparable adsorption behaviour of gelatin has been observed in literature [2,18], often in conjunction with other surfactants (e.g. [7,19]). The adsorp-
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gelatin concentration on a double logarithmic scale. A linear relationship is observed, a behaviour which has been also observed for other polymeric surfactants [6,16]. The induction time scales with 1/Cn . The slope, n, of the concentration dependence of the induction phase of gelatin 65B (Fig. 5a) is steeper than the one of gelatin 280B (Fig. 5b). There is a large average size difference between both samples. There might be cooperative effect facilitating the attachment at the interface of more polymer subunits, when there is already a part of the molecule adsorbed. The cooperative effect would be higher for the larger chains. Actually, it might be easier to adsorb one subunit of the same polymer chain than to adsorb a new chain. The hypothesis that diffusion governs the adsorption of surfactants at the air/water interface is largely accepted in literature. Lin et al. [7] showed that a characteristic time scale of diffusion ( d ) for gelatin at room temperature could be estimated according the Frumkin isotherm. In the dilute regime one has d ≈
Fig. 4. Development of the surface pressure of gelatin 65B at different bulk concentrations in acetate buffer (a) and water (b) at 42 ◦ C. Symbols: 1 wt% (), 0.1 wt% (), 1 × 10−2 wt% (♦), 1 × 10−3 wt% (*), 1 × 10−5 wt% (+).
tion curves plotted in Fig. 4, show that there are two different behaviours: a first adsorption step which is too fast to be detected by our measuring set-up for higher concentrations (0.1 and 1.0 wt%), and a second, slower phenomenon which is linear on the logarithmic time scale until the adsorption reaches the vicinity of the equilibrium surface pressure. We attribute the initial surface pressure (˘ i ) higher than zero to an artefact. Indeed, the formation of the bubble in the measuring device takes approximately one second during which adsorption of gelatin already occurs for the higher concentrations. However, the measuring of the surface tensions starts only once the bubble formation is completed. Moreover, we suppose that there is most probably interference in the first values due to the oscillation of the bubble shape caused by the elasticity of the medium. In Fig. 1 is shown that there is a large uncertainty on the initial curve values. It is however interesting to compare the height of ˘ i between both samples. Actually, the values of sample 280B are systematically lower than the values of sample 65B (8 mN/m for gelatin 65B versus 4 mN/m for gelatin 280B, both at 0.1 wt% in acetate buffer). Taking into account that gelatin is polymolecular, it is suggested that there is roughly a population of smaller chains which colonized the surface faster, and a population of larger chains contained in both samples. The smaller chains are more abundant in gelatin 65B, having the smaller [] which leads to a higher ˘ i . The rate of attachment, being faster for smaller chains, governs also the length of the induction phase in the case for which ˘ i = 0. Fig. 5 shows the numerical values of the induction time versus the
Fig. 5. Relationship between the lag time (tlag ) and the gelatin concentration at 42 ◦ C. Sample 65B is plotted in (a) and sample 280B in (b). Symbols: gelatin 65B in acetate buffer (), gelatin 65B in water (䊉), gelatin 280B in acetate buffer (), gelatin 280B in water ().
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Fig. 6. Variation of the differential of the surface pressure (d˘/dt) with time for gelatin 65B in water and in acetate buffer (a) and gelatin 280B in acetate buffer and in water (b). Symbols: 1 wt% (), 0.1 wt% (), 1 × 10−2 wt% (♦), 1 × 10−3 wt% (*), 1 × 10−4 wt% (+), 1 × 10−5 wt% (×).
2
(∞ e˛/kB T /C) /D, where ∞ is the surface coverage (∼2 mg/m2 , Marshall 2002 [19]), C the bulk concentration, and D is the diffusion coefficient (in the order of 10−12 m2 /s, estimation of Lin et al. at room temperature [7]). The authors show furthermore that the surface activity of gelatin in solution is quite weak, thus one can assume ˛/kB T ≈ 1. In our case, d would be in the order of 4 e2 s (∼30 s) for a concentration of 0.1 wt% and about 400 e2 (∼3000 s) for 0.01 wt%. The adsorption would thus be completed within one minute for the higher concentrations, and within one hour for the smaller ones. However, we see in Fig. 4 that the equilibrium surface pressure is not obtained within this timeframe. Beyond the initial diffusion-limited state a slower relaxation phenomenon is seen leading to a further increase of the surface pressure has been associated to a slow molecular reorganization process [7,15]. In order to reveal some physical information about the adsorption process, we analyze the rate of surface pressure increase with the help of the treatment published by Millet et al. [16]. Fig. 6 plots the time dependency of the rate of the surface pressure increase (d˘/dt). Fig. 6a shows the results for gelatin 65B in acetate buffer and in water, and Fig. 6b for gelatin 280B in water and acetate buffer. We observe a first step during which (d˘/dt) changes merely. However, data are quite noisy. For a more clear presentation, the very noisy initial phase was cut for the different curves. More important, subsequent to this phase, all curves
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fall on the same line being linear with 1/t. The slope of (d˘/dt) versus time amounts approximately to −1 in all cases studied, i.e. gelatin 65B and gelatin 280B in acetate buffer or water. Once the air/water interface is covered with gelatin, there is a continuous slowing down of the rate of the surface pressure increase, which is not correlated to the bulk properties. Such logarithmic kinetics is a sign of self-inhibited systems. Millet et al. [16] interpret the behaviour on the basis of a theoretical model developed by Ligoure and Leibler [20] for neutral diblock copolymers from specific solvents. At times larger than the time to reach a critical surface pressure, adsorbed chains form a dense brush creating steric hindrance to further adsorption in the form of dangling chains in the subsurface. According to the model, the diffusion of free chains in the solvent does not control the adsorption kinetics. Once the brush has formed, additional chains have to stretch out in the vicinity of the interface and adopt a reptation-diffusion mechanism in order to penetrate the brush. The authors show that in this case a logarithmic variation of the surface excess amount of molecules ( ) with time is obtained. To compare to the experimental data, a relationship between and ˘ has to be obtained. With the help of the general expression ˘ ∝ n , where n expresses the type of interaction predominant in the adsorption layer, the slope of the curve (d˘/dt) versus time can be computed. It continuously decreases with time, approaching asymptotically −1 in the limit of infinite time. The mathematical treatment is given by Millet et al. [16]. The slopes calculated for the different samples measured here show the value of −1 within the experimental uncertainty. The adsorption of gelatin to the air/water interface can thus reasonably be interpreted within the framework of this theory, although the chemical structure of the gelatin molecule is rather far from a neutral diblock copolymer with a sharp molar weight distribution. However, we suppose that there is the formation of an encumbered surface layer with dangling gelatin chains into the solvent. Additional evidence for the formation of an encumbered interface sub-layer might come from the direct measurement of the thickness of interfacial films. Observation of the surface layer of gelatin by X-ray reflectivity at 40 ◦ C showed that there are two types of dense regions with a layer thickness of 14 Å and an extended region of 94 Å containing only a small percentage of gelatin (5 vol%). This gives a layer thickness of approximately 108 Å, having very high roughness [21]. The surface layers measured with this technique are smaller than the ones from other techniques, because X-ray reflectivity cannot resolve extended chains in the bulk. The surface coverage measured is 1.7 mg/m2 . The surface layer of gelatin on the polystyrene/water interface was measured by small angle neutron scattering (SANS). The layer thickness was approximately 130 Å with a surface coverage of 1.9 mg/m2 [19]. Those layers are still thin compared to the thickness measured in ellipsometry on hydrophobic silica surfaces, amounting to 400 Å (values taken from Ref. [19]). Gelatin is a polyampholyte. Therefore it is capable to develop cationic, anionic and hydrophobic interactions, having binding sites irregularly arranged. This allows the molecules to associate with the interface by electrostatic and/or hydrophobic interactions. We observe with the help of the analysis of the viscosity a large influence of the salt concentration of the solvent on the chain expansion. However, the theory presented here was developed for neutral diblock copolymers. Therefore, we have supposed implicitly that electrostatic interactions can be neglected in our case. Actually, the (d˘/dt) curves regroup to a single curve for one sample, no matter the solvent and the kinetics, and are unaffected by the salt concentration (Fig. 6). The adsorbed layer is probably self-screened due to interactions between the gelatin molecules, so that no repulsing
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ics on the air/water surface were done in the dilute regime. The analysis of the kinetics of the surface pressure increase leads us to the following conclusions:
Fig. 7. Relationship between the equilibrium surface pressure and the gelatin concentration at 42 ◦ C. The lines are drawn to guide the eye. Symbols: gelatin 65B in acetate buffer (), gelatin 65B in water (䊉), gelatin 280B in acetate buffer (), gelatin 280B in water ().
effect between adsorbed layer and arriving molecules is detected, which would cause a delay in adsorption kinetics. Furthermore, we observe no effect of the chains size of gelatin comparing the (d˘/dt) versus time curves for both samples. Comparing the axis of Fig. 6a and b, we see that there is actually no delay between the curves within the experimental uncertainty. One might have expected a delay in the (d˘/dt) versus time curve to longer times for samples with higher molecular size. We think that this is not observed due to the polymolecular character of gelatin, which contains in both samples longer and smaller chains. Fig. 7 plots the results of the equilibrium surface pressure versus gelatin concentration for both samples in both solvents. There is no difference between the equilibrium values for the 280B gelatin in function of the solvent within the experimental uncertainty (±2 mN/m), suggesting that there is no influence of salt concentration on the adsorption and structuring of 280B gelatin on the interface. The equilibrium surface pressure results of the 65B gelatin in water seem to be more stretched on the ordinate axis direction, resulting in low equilibrium surface pressures at low concentrations and higher values at high concentrations. We attribute the behaviour to the polymolecularity of the gelatin samples, containing also branched chains. The sample 65B contains a higher proportion of smaller gelatin chains which might remain more flexible at the interface and thereby allow a higher surface coverage at higher bulk solutions. 4. Conclusion The behaviour of two gelatin samples in solution and at the air/water interface was investigated at 42 ◦ C in order to ensure the coil conformation of the protein molecules. We observe an expansion of gelatin upon dilution in water due the polyelectrolyte nature of the protein chain. The increase of ionic strength of the solvent reduces successfully the expansion. We calculated an apparent value of the intrinsic viscosity of both samples which could be successfully used to plot a master curve of the development of the specific viscosity of both samples on a reduced concentration scale. According to the critical overlap concentration all adsorption kinet-
(1) The analysis of the adsorption kinetics revealed differences between both gelatin samples in terms of the length of the induction phase and the dependency of the equilibrium pressure on the sample concentration. We interpret that there are roughly two populations of gelatin molecules contained in the samples, one having small and the other having long chain length, which are relevant for the adsorption behaviour. The smaller chains adsorb faster to the interface bringing about smaller induction times. Therefore gelatin 65B, being more hydrolyzed, had smaller induction periods. However, at very small gelatin concentrations (10−5 wt%) the sample being composed of mainly larger chains showed the smaller induction time. This effect might be due to a cooperative effect of the long chains, where the adsorption of the subunits of an already attached chain might be easier than the adsorption of a new chain. A second effect of the polymolecular character of the gelatin samples was that the more hydrolyzed sample showed on one hand higher equilibrium surface pressures at high bulk concentrations than the less hydrolyzed sample, but on the other hand smaller equilibrium surface pressures at small bulk concentrations. We think that the same cooperative effect caused this behaviour at small concentrations. (2) The analysis of the adsorption kinetics showed a continuous slow down of the rate of surface pressure increase after the initial adsorption phase, which is linear on a logarithmic time scale with a slope of −1. This behaviour is characteristic for self-inhibited systems. It might be caused by the formation of an encumbered subsurface layer which causes steric hindrance of the newly arriving chains. The adsorption kinetics is analogous to the kinetics of surface-active copolymers, which form a brush on the interface with dangling ends in the solution. Our interpretation might be supported by the observation of surface film layers with high thickness by direct methods, such as ellipsometry or SANS. (3) The adsorption kinetics seems to be independent from the salt concentration of the solvent, although the ionic strength had a high impact on the expansion of the gelatin chains. Once the first, diffusive phase, accomplished, the adsorption kinetics are independent of the gelatin chain length, a behaviour observed also for other surface active polymers.
References [1] S. Damodaran, Interfaces, protein films, and foams, Adv. Food Nutr. Res. 34 (1990) 1–79. [2] H. Sato, K. Ueberreiter, Surface tension of aqueous gelatin solutions, 1, concentration dependence, Maromol. Chem. 180 (1979) 829–835. [3] N. Kitabatake, E. Doi, Surface tension and foaming of protein solutions, J. Food Sci. 47 (1982) 1218–1221. [4] V. Galazka, E. Dickinson, Surface properties of protein layers adsorbed from mixtures of gelation with various caseins, J. Text. Stud. 26 (1995) 401– 409. [5] A. Mackie, P. Gunning, M. Ridout, V. Morris, Gelation of gelatin: observation in the bulk and at the air–water interface, Biopolymers 46 (1998) 245– 252. [6] T. Gilanyi, M. Varga, M. Gilanyi, R. Meszaros, Adsorption of poly(ethylene oxide) at the air/water interface: a dynamic and static surface tension study, J. Colloids Interf. Sci. 301 (2006) 428–435. [7] S.-Y. Lin, T.-F. Wu, H.-K. Tsao, Interfacial dynamics of a gelatin solution with surfactant, Macromolecules 36 (2003) 8786–8795. [8] T. Kravtchenko, W. Pilnik, A simplified method for the determination of the intrinsic viscosity of pectin solutions by classical viscosimetry, Gums Stabil. 5 (1990) 281–285. [9] C.H. Lee, C.S. Kim, H.C. Yang, Microstructure and hydrodynamic properties of soybean protein bodies in solutions, J. Food Sci. 48 (1983) 695–702.
S. Domenek et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 331 (2008) 48–55 [10] C.T. Andrade, E.G. Azero, L. Luciano, M.P. Gonc¸alves, Solution properties of the galactomannans extracted from the seeds of Caesalpinia pulcherrima and Cassia javanica: comparison with locust bean gum, Int. J. Biol. Macromol. 26 (1999) 181–185. [11] G. Cuvelier, B. Launay, Concentration regimes in xanthan gum solutions deduced from flow and viscoelastic properties, Carbohyd. Polym. 6 (1986) 321–333. [12] G. Robinson, S.B. Ross-Murphy, E.R. Morris, Viscosity–molecular weight relationships, intrinsic chain flexibility, an dynamic solution properties of guar galactomannane, Carbohydr. Res. 107 (1982) 17–32. [13] M.R. Rodrigez-Nino, C. Carrera-Sanchez, V. Pizones Ruiz-Henestrosa, J.M. Rodriguez-Patino, Milk and soy protein films at the air–water interface, Food Hydrocolloids 19 (2005) 417–428. [14] A.F.H. Ward, L. Tordai, Time-dependence of boundary tensions of solutions. I. The role of diffusion in time-effects, J. Chem. Phys. 14 (1946) 453–461. [15] A.M. Diez-Pascual, A. Compostizo, A. Crespo-Colin, R.G. Rubio, R. Miller, Adsorption of water-soluble polymers with surfactant character. Adsorption kinetics and equilibrium properties, J. Colloid Interf. Sci. 307 (2007) 398–404.
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[16] F. Millet, P. Perrin, M. Merlange, J.J. Benattar, Logarithmic adsorption of charged polymeric surfactants at the air–water interface, Langmuir 18 (2002) 8824–8828. [17] S. Mezdour, G. Cuvelier, M.J. Cash, C. Michon, Surface rheological properties of hydroxypropyl cellulose at air/water interface, Food Hydrocolloids 21 (2007) 776–781. [18] H. Sato, K. Ueberreiter, Surface tension of aqueous gelatin solutions, 2, the effects of pH and temperature, Maromol. Chem. 180 (1979) 1107–1112. [19] J.C. Marshall, T. Cosgrove, K. Jack, Small-angle neutron scattering of gelatin/sodium dodecyl sulphate complexes at the polystyrene/water interface, Langmuir 18 (2002) 9668–9675. [20] C. Ligoure, L. Leibler, Thermodynamics and kinetics of grafting endfunctionalized polymers to an interface, J. Phys. France 51 (1990) 1313– 1328. [21] K.A. Vaynberg, N.J. Wagner, H. Ahrens, C.A. Helm, Gelatin adsorption at the air/water interface as investigated by X-ray reflectivity, Langmuir 15 (1999) 4685–4689.
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Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Influence of concentration and ionic strength on the adsorption kinetics of gelatin at the air/water interface S. Domenek a,∗ , E. Petit a , F. Ducept a , S. Mezdour a , N. Brambati b , C. Ridoux b , S. Guedj b , C. Michon a a b
UMR Ingénierie Procédé Aliment, AgroParisTech-INRA-CNAM-CEMAGREF, 1 avenue des Olympiades, 91744 Massy Cedex, France Rousselot, Chemin Moulin Premier, 84808 Isle sur la Sorgue Cedex, France
a r t i c l e
i n f o
Article history: Received 31 January 2008 Received in revised form 11 June 2008 Accepted 13 June 2008 Available online 22 June 2008 Keywords: Gelatin Intrinsic viscosity Adsorption kinetics Air/water interface Foam
a b s t r a c t The adsorption kinetics of gelatin to the air/water interface is still not fully understood. We investigated two samples of gelatin having different gel strength (65 Bloom and 280 Bloom), named, respectively gelatin 65B and 280B. The gelatin samples in solution were characterised by viscosimetric methods. We evidence a chain expansion of gelatin upon dilution due to the polyelectrolyte effect. The measurement of the intrinsic viscosity in water gave values of 22 mL/g for the sample 65B and 70 mL/g for the sample 280B. Although gelatin is a highly polymolecular polymer containing branched chains, the normalisation of the concentration by the intrinsic viscosity allowed the construction of a master curve of viscosity, giving the reduced critical overlap concentrations c* ∼ 0.6 and c** ∼ 5. All concentrations of the gelatin solutions used for the determination of the adsorption behaviour were within the range of the dilute regime. The analysis of the adsorption behaviour leads us to the hypothesis that there are two different phenomena: first, a step governed by diffusion to the interface which lasts until full coverage of the interfacial film. The length of this induction period seemed to be dependent on the gelatin chain length, with larger chains taking more time. Second, the adsorption of the chains to the air/water interface leads to the formation of an encumbered subsurface layer which opposes steric hindrance to newly arriving chains. This causes a continuous slow down of the rate of surface pressure increase, which was linear on the logarithmic time scale and showed a slope of −1. The adsorption kinetics was independent of the salt concentration of the solvent and of the gelatin chain size measured by the intrinsic viscosity. The gelatin sample 65B shows furthermore higher equilibrium surface pressures at high bulk concentrations (>0.01 wt%) than sample 280B, while the contrary was observed at small concentrations. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Gelatin is widely used in food industry for the stabilisation of foamed products because of its surface-active and gelling upon cooling. For the formulation of foamed products it is important to understand the behaviour of gelatin at the air/water interface. Foaming of gelatin solutions is often done at thermal conditions higher than the gelling temperature. In this case differences between gelatin samples can be observed. In order to discriminate the effects of the surface activity of gelatin from its gelling properties, it is necessary to study its interfacial behaviour in nongelling conditions. The adsorption onto the air/water interface of heteropolymers containing hydrophobic segments, such as pro-
∗ Corresponding author. Tel.: +33 1 69 93 50 68; fax: +33 1 69 93 50 05. E-mail address: [email protected] (S. Domenek). 0927-7757/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2008.06.034
teins, is a very much slower process than the adsorption of small surfactants owing to the much greater size and structural complexity. In the case of macromolecules, it may take several hours to reach the equilibrium. As a consequence, the time dependent interfacial properties play an important role in industrial processes. Gelatin, the hydrolysis product of structural collagen, has a very particular structure among proteins. It presents a high structural complexity because it has a large size distribution of the protein chains which are mainly linear although there is a small percentage non-hydrolyzed bonds giving rise to branched chain. However, it has no well-defined spatial conformation like globular proteins. It can thus also be considered as an ampholytic polymer with an isoelectric point varying between pH 7.5 and 9.3 for type A gelatin and 4.7 and 5.2 for type B gelatin. At temperatures higher than 40 ◦ C it has a loosely packed coil structure. Upon cooling at less than 40 ◦ C, the gelatin chains can undergo a conformation change and arrange themselves in triple helices, which might change the foaming behaviour of gelatin.
S. Domenek et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 331 (2008) 48–55
Generally, the adsorption behaviour of proteins on the interfaces is described as a diffusive process with an energy barrier, including a phase of attachment of the proteins on a cleared interface area and a phase of rearrangement and denaturation in the adsorbed layer [1]. Sato and Ueberreiter [2] studied the adsorption of gelatin at the air/water interface at 40 ◦ C for different concentrations. They conclude that the surface concentration of gelatin is approximately 14-fold higher than the concentration in the bulk solution. Depending on the concentration of gelatin, the authors define different degrees of intermolecular interactions of gelatin molecules on the surface. At low surface concentrations there seemed to be a higher structuring of the gel than at higher concentrations (above 0.25 mg/mL). Several studies have been published on the behaviour of gelatin at the air/water interface, where the solutions were kept at room temperature. In this case, the gelatin molecules are able to form triple helices. The rate constants of the adsorption kinetics were calculated with the help of a first order exponential law analogous to the Avrami equation, which has the inconvenience that the equilibrium surface tension intervenes strongly in the calculation, which is generally difficult to determine experimentally. The relationship between foam volume and adsorption kinetics of various proteins was studied at room temperature [3]. A single, linear relationship between foam volume and rate constants was observed for different globular proteins. Gelatin, however, showed a distinct behaviour, having, relatively to the other proteins, a slow adsorption rate, but a high foam volume. This behaviour was attributed to its molecular properties and the solution viscosity being different from globular proteins [3]. The formation of a gel structure at the interface by gelatin has been evidenced by the measurement of the shear viscosity of gelatin/␣-casein mixtures at the interface [4], and by investigation of thin gelatin films with the help of atomic force microscopy [5]. The structural parameters playing obviously a role in the surface activity, it has been shown for surface active polymers such as poly(ethylene oxide) that the equilibrium surface concentration of the polymer, expressed in mol/m2 , diminished with an increase of molecular weight. However, for a polymer chain length higher than 2 × 103 the surface concentration became independent of the bulk concentration [6]. Lin et al. [7] investigated the interfacial dynamics of gelatin solutions together with different surfactants at 25 ◦ C when gelatin is in helix conformation. They showed that there was a long relaxation time leading to a gradual increase of the interfacial tension which was attributed to intraand interchain rearrangements of the gelatin chains occurring at the interface. Although there are data and descriptions of kinetics of the gelatin adsorption behaviour published, only Lin et al. [7] proposed a possible mechanism to understand the adsorption behaviour. For their analysis they considered gelatin being a linear polymer with a molar weight between 1 and 2 × 105 . It is commonly admitted that the first step of the polymer attachment on the interface is diffusion controlled. They showed, furthermore, that gelatin had a long relaxation time signature, which they attributed to the intra- and interchain rearrangements of the molecules at the interface may be a process analogous to the gel formation in bulk solution. Foaming is a dynamic process. Therefore, it is interesting to investigate the adsorption kinetics in further detail. The understanding of the kinetics can help to get more insight into the physical mechanisms of the adsorption process. Because industrial foaming processes are often carried out at temperatures higher than the conformation change (at 40 ◦ C) leading to the gel formation by gelatin, we worked here at temperatures higher than 40 ◦ C, in order to keep gelatin in its coil conformation. We used two different gelatin samples, distinguished by their gel strength. The viscosity of the gelatin solutions was investigated in order to determine the limits of the different concentration regimes. The
49
adsorption kinetics of both samples was subsequently measured at different concentrations in the dilute regime. Two solvents were used differentiated by their salt content, but not by their pH in order to investigate the polyelectrolyte behaviour of gelatin samples. The kinetics of the recorded surface tension was analyzed. 2. Materials and methods All chemicals were purchased from Sigma–Aldrich (France). The gelatin samples (type A, acid hydrolysis) were provided by Rousselot SA (France). Sample 65B and 280B have gel strengths of 65 and 280 Bloom, respectively. The pH of both gelatin samples after dissolution in water was between 4.8 and 5, their isoelectric point being at approximately pH 8.5. Solutions were prepared in dionised water or in an acetate buffer (pH 5, 0.2 mol/L) in order to control the ionic strength of the solution. The mineral contents of sample 65B and 280B were 16.7 and 5.8 mg/g, respectively corresponding to, in 1 wt% gelatin solutions prepared in deionised water, at most about 2 × 10−5 and 0.7 × 10−5 mol/L of free ions. These extremely low ion concentrations correspond to less than 0.03% of the ionic strength of the buffer solution which can be neglected. Moreover, they can be also reasonably neglected in deionised water solutions when studying concentrations below 1 wt%, in the diluted regime. In order to melt the triple-helix aggregates of gelatin, the solutions were heated at 50 ◦ C for 30 min prior to the measurements. All solutions were prepared freshly each 3 days and stored at 4 ◦ C. The determination of the viscosity of the gelatin samples in the low concentration range at 44 ◦ C was carried out using a capillary viscosimeter equipped with an automatic dilution system (Viscologic, Laser Diffusion Systems, France). The determination of each time of passage through the capillary was done in 10 replicates and each solution was at least duplicated. The determination of the viscosity of the gelatin solutions at higher concentrations (≥0.1 wt%) was realised using a Rheometrics Fluid Rheometer (RFS II, TA Instrument) working with imposed deformation. The instrument was fitted with coaxial cylinders (R1/R2 = 0.96). A flow curve was carried out in the shear rate range 0.1–150 s−1 . The viscosity value was taken in the Newtonian plateau. The reduced viscosity (red ) of the samples was calculated after red =
− solvent 1 solvent C
(1)
where is the measured viscosity, solvent the viscosity of the solvent (water or acetate buffer), and C is the concentration (g/100 mL). Each concentration was measured in triplicate. The density of the diluted solutions was equal to water. Therefore we considered the weight percentage in the dilute regime equal to the unit g/100 mL. For concentrations higher than 1 g/100 mL we worked in wt%. The measurement of the surface tension of the gelatin solutions was carried out with an automated tensiometer (Tracker, IT Concept, France). The principle of the measurement is based on axisymmetric drop shape analysis of an air bubble at the end of a capillary in the solution containing the surfactant. The surface tension is calculated with the help of the Laplace equation. The bubble volume was 9 L and the temperature of the gelatin solution was maintained at 42 ◦ C with the help of a sample holding device equipped with double wall jacket and branched to a water bath. The temperature of the solution and the surface tension were recorded online. In order to avoid the evaporation of the solution a layer of paraffin oil was placed on the surface of the gelatin solution. The non-miscibility of water and paraffin oil and the non-perturbation of the measurement were verified during several hours with deionised water. The surface tension of water and the acetate buffer were checked each day. In the following the decrease of the surface tension is expressed as an increase of surface pressure
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Fig. 1. Reproducibility of the adsorption kinetics for four repeat experiments of gelatin 65B at 0.1 and 0.001 wt% in acetate buffer.
( = solvent − gelatin ) in order to normalise small variations in the solvent surface tension. Fig. 1 shows the reproducibility of the evolution of the surface pressure versus the logarithmic time scale for one gelatin sample (65B) at two concentrations. We observe that the experimental error on the data is larger at short times, where we have an uncertainty of 2 mN/m, and the curves at zero surface pressure are noisy. However, the overall shape of curves is reproducible and the end values show smaller error. Due to the long experiment time required to approach equilibrium values (>10 h) and the sensibility of the experiment to external factors, the equilibrium value was not approached for each experiment. For analysis, the following parameters were quantified: (i) the initial surface pressure (˘ i ) read at time zero, the length of the induction phase (tlag ) quantified by the calculation of the crossing point between the initial straight regression line and the tangent to the surface pressure increase, and the value of the equilibrium pressure (˘ eq ) calculated with the help of a sigmoid line fit (logistic function with four parameters), in the case that the beginning of the final plateau was observed. The quality of the fit was tested on the surface tension curves, where a final plateau value was observed. For differentiation, curves were interpolated on the logarithmic time scale with 90 points and a moving average was calculated over 10 points. The derivative was filtered by suppressing negative and zero values. Computing was done with Matlab v6 software (Matworks Inc.).
Fig. 2. Evolution of the reduced viscosity (red ) of the gelatin solutions versus concentration at two salt concentrations. Symbols: gelatin 65B in acetate buffer (), gelatin 65B in water (䊉), gelatin 280B in acetate buffer (), and gelatin 280B in water ().
determination yields an apparent average value being an average of the gelatin chains contained in the sample. Fig. 2 shows the evolution of the reduced viscosity with the concentration in both solvents, water and acetate buffer at the same pH for the two gelatin samples. The 280B sample has a higher reduced viscosity than the 65B sample, meaning that it contains in average larger molecules, which is consistent with its higher Bloom value. Both samples show an increase of the reduced viscosity upon dilution in water, which is a typical behaviour of polyelectrolytes. The increase of the ionic strength of the solvent (acetate buffer 0.2 mol/L) without change of the pH of the gelatin solutions decreases successfully the expansion confirming the polyelectrolyte effect hypothesis. However, even in the acetate buffer solvent, the small remaining expansion of gelatine chains in diluted conditions prevents us from extrapolating the reduced viscosity to zero gelatin concentration using the Huggins equation. The intrinsic viscosity was, thus, calculated using the relationship developed by Kravtchenko and Pilnik [8] for polyelectrolytes, which allows the extrapolation to [] from the plateau of the reduced viscosity at higher concentrations. The intrinsic viscosity was computed from the gelatin concentration (C) of 0.5 wt% using Eq. (2): [] =
3. Results and discussion 3.1. Determination of the intrinsic viscosity and the critical overlap concentration In order to characterise the gelatin samples, we studied the viscosity of the bulk solution. The ability to increase solution viscosity at a given concentration of polymers is dependent on the macromolecular size, rigidity and degree of branching, which contribute both to its hydrodynamic volume. Gelatin develops high solution viscosities compared to other proteins due to its flexibility and, thus, to its expanded coil structure. A method to determine the hydrodynamic volume of a polymer chain is the measurement of its intrinsic viscosity ([]), i.e. its hydrodynamic volume in diluted conditions. Because gelatin is a highly polymolecular polymer, the
1 2 C
solvent
− 1 − ln
12
(2)
solvent
The results are given in Table 1. The values of [] are much higher than those observed for globular proteins, such as soybean protein (0.05 dL/g [9]), but considerably lower than the intrinsic viscosity of polysaccharides used for stabilisation purposes in the food industry, such as locust bean gum (15 dL/g [10]) or xanthan (49.3 dL/g [11]). A marked difference in the average hydrodynamic volume is observed Table 1 Apparent intrinsic viscosity [] of the gelatin samples calculated after Kravtchenko and Pilnik [8]
Gelatin 65B Gelatin 280B
[] (dL/g) Acetate buffer
[] (dL/g) Water
0.22 ± 0.01 0.50 ± 0.01
0.27 ± 0.01 0.70 ± 0.07
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the polyelectrolyte effect, which increases the viscosity at small concentrations due to the chain expansion. Fig. 3b shows that the reduction of the concentration axes by the apparent intrinsic viscosity value yields a good adjustment of both samples curves to a single master curve, although gelatin chains have a broad size distribution. The transition zone between diluted and concentrated dilution regime stretches from 0.6 to 5 on the reduced concentration scale (Fig. 3b). The slopes are lower than the slopes observed for other biopolymers in coil conformation. For example, guar gum yields a slope of 1.2 in the dilute regime, 2.4 in the semi-dilute, and 5.4 in the concentrated regime [12], and the more rigid xanthan gum 1.25, 2.1, and 4.2, respectively [11]. Gelatin, in comparison to polysaccharides, is composed of more flexible chains. This flexibility brings about a higher capacity of expansion or contraction of the chains in function of their concentration in solution. In order to investigate whether the chain expansion of gelatin had an influence on the adsorption mechanism on the interface and in order to be able to detect the initial evolution of the surface pressure when gelatine starts to adsorb, we choose to work with both samples in the dilute concentration regime. 3.2. Development of the surface pressure in function of the gelatin concentration and the ionic strength
Fig. 3. Relationship between reduced viscosity and gelatin concentration. Reduced viscosity versus bulk concentration (a), reduced viscosity versus the bulk concentration scale reduced with the intrinsic viscosity calculated after Kravtchenko and Pilnik [8] (b). Symbols: gelatin 65B (䊉) and 280B () dissolved in water.
between both gelatin samples. The volume decrease of gelatin 280B chains in acetate buffer compared to water is much higher than the one of gelatin 65B. Generally, the larger the chain length of a polymer, the higher is its expansion ability. In order to specify the limits of the dilute regime of the gelatin solutions, the reduced viscosity was measured in a larger concentration range for both samples dissolved in water. This treatment allowed choosing the concentration range for the following tensiometric measurements. The results are shown in Fig. 3a. In concordance with [] difference, the change from the dilute to the concentrated regime is shifted to higher concentrations for sample 65B. A critical overlap concentration of approximately 3 wt% for gelatin 65B and 1.5 wt% for gelatin 280B is read (Fig. 3a). The slopes of the reduced viscosity–concentration curve in the dilute regime are smaller than slopes generally found for other polymers. Andrade et al. [10] measured 1.2 for locust bean gum in the dilute regime. The measured slope is most likely underestimated due to
The rate of surface occupation and the ability to develop a high surface pressure of a surface-active macromolecule are of importance in the foaming process. In order to study the phenomenon and to deepen our understanding of the adsorption process, the kinetics of the adsorption of gelatin to the air/water interface was investigated in a concentration range from 1 × 10−5 to 1 wt%. In Fig. 4 the increase of the surface pressure of gelatin 65B in acetate buffer and water is plotted on the logarithmic time scale as an example. All curves show a sigmoid shape with an induction phase. It follows a steep increase in surface pressure and a final phase approaching the thermodynamic equilibrium between the adsorbed layer and the bulk solution. The adsorption behaviour of globular proteins shows generally a comparable shape, although the induction phase is rarely observed for proteins. This is mainly due to the utilisation of high protein concentrations. For smaller concentrations, lag-phases can be observed [13]. Generally, the adsorption kinetics is described by an exponential decay [1,3]. The interpretation of the curve shape proposed is an induction phase due to diffusion to the interface, an adsorption phase of the proteins on the interface involving penetration and anchoring on a cleared interface area, and a phase of slow rearrangement of the molecules at the interface related to reorientation and conformational change [1]. In many adsorption studies on proteins, the rates of adsorption have been shown to be lower than the rate of diffusion [1]. In order to account for this behaviour, it was proposed that there is an activation energy existing at the interface for adsorption of the solute from the subsurface [14]. This energy barrier might be related to the denaturation of the proteins at the surface. Gelatin however, being already a denatured product of collagen, has no defined special conformation such as globular proteins. Moreover, in our experiments the gelatin solutions were kept at 42 ◦ C which keeps the molecules in their coil state. Therefore, gelatin could be considered as a linear polymer including a small number of branched chains. The form of the adsorption curve plotted in Fig. 4 is classically observed for the adsorption of polymer surfactants to interfaces, such as poly(ethylene oxide) [6], copolymers of poly(propylen glycol) [15], grafted poly(acrylic acid) [16], or biopolymers such as hydroxypropyl cellulose [17]. Furthermore, comparable adsorption behaviour of gelatin has been observed in literature [2,18], often in conjunction with other surfactants (e.g. [7,19]). The adsorp-
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gelatin concentration on a double logarithmic scale. A linear relationship is observed, a behaviour which has been also observed for other polymeric surfactants [6,16]. The induction time scales with 1/Cn . The slope, n, of the concentration dependence of the induction phase of gelatin 65B (Fig. 5a) is steeper than the one of gelatin 280B (Fig. 5b). There is a large average size difference between both samples. There might be cooperative effect facilitating the attachment at the interface of more polymer subunits, when there is already a part of the molecule adsorbed. The cooperative effect would be higher for the larger chains. Actually, it might be easier to adsorb one subunit of the same polymer chain than to adsorb a new chain. The hypothesis that diffusion governs the adsorption of surfactants at the air/water interface is largely accepted in literature. Lin et al. [7] showed that a characteristic time scale of diffusion ( d ) for gelatin at room temperature could be estimated according the Frumkin isotherm. In the dilute regime one has d ≈
Fig. 4. Development of the surface pressure of gelatin 65B at different bulk concentrations in acetate buffer (a) and water (b) at 42 ◦ C. Symbols: 1 wt% (), 0.1 wt% (), 1 × 10−2 wt% (♦), 1 × 10−3 wt% (*), 1 × 10−5 wt% (+).
tion curves plotted in Fig. 4, show that there are two different behaviours: a first adsorption step which is too fast to be detected by our measuring set-up for higher concentrations (0.1 and 1.0 wt%), and a second, slower phenomenon which is linear on the logarithmic time scale until the adsorption reaches the vicinity of the equilibrium surface pressure. We attribute the initial surface pressure (˘ i ) higher than zero to an artefact. Indeed, the formation of the bubble in the measuring device takes approximately one second during which adsorption of gelatin already occurs for the higher concentrations. However, the measuring of the surface tensions starts only once the bubble formation is completed. Moreover, we suppose that there is most probably interference in the first values due to the oscillation of the bubble shape caused by the elasticity of the medium. In Fig. 1 is shown that there is a large uncertainty on the initial curve values. It is however interesting to compare the height of ˘ i between both samples. Actually, the values of sample 280B are systematically lower than the values of sample 65B (8 mN/m for gelatin 65B versus 4 mN/m for gelatin 280B, both at 0.1 wt% in acetate buffer). Taking into account that gelatin is polymolecular, it is suggested that there is roughly a population of smaller chains which colonized the surface faster, and a population of larger chains contained in both samples. The smaller chains are more abundant in gelatin 65B, having the smaller [] which leads to a higher ˘ i . The rate of attachment, being faster for smaller chains, governs also the length of the induction phase in the case for which ˘ i = 0. Fig. 5 shows the numerical values of the induction time versus the
Fig. 5. Relationship between the lag time (tlag ) and the gelatin concentration at 42 ◦ C. Sample 65B is plotted in (a) and sample 280B in (b). Symbols: gelatin 65B in acetate buffer (), gelatin 65B in water (䊉), gelatin 280B in acetate buffer (), gelatin 280B in water ().
S. Domenek et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 331 (2008) 48–55
Fig. 6. Variation of the differential of the surface pressure (d˘/dt) with time for gelatin 65B in water and in acetate buffer (a) and gelatin 280B in acetate buffer and in water (b). Symbols: 1 wt% (), 0.1 wt% (), 1 × 10−2 wt% (♦), 1 × 10−3 wt% (*), 1 × 10−4 wt% (+), 1 × 10−5 wt% (×).
2
(∞ e˛/kB T /C) /D, where ∞ is the surface coverage (∼2 mg/m2 , Marshall 2002 [19]), C the bulk concentration, and D is the diffusion coefficient (in the order of 10−12 m2 /s, estimation of Lin et al. at room temperature [7]). The authors show furthermore that the surface activity of gelatin in solution is quite weak, thus one can assume ˛/kB T ≈ 1. In our case, d would be in the order of 4 e2 s (∼30 s) for a concentration of 0.1 wt% and about 400 e2 (∼3000 s) for 0.01 wt%. The adsorption would thus be completed within one minute for the higher concentrations, and within one hour for the smaller ones. However, we see in Fig. 4 that the equilibrium surface pressure is not obtained within this timeframe. Beyond the initial diffusion-limited state a slower relaxation phenomenon is seen leading to a further increase of the surface pressure has been associated to a slow molecular reorganization process [7,15]. In order to reveal some physical information about the adsorption process, we analyze the rate of surface pressure increase with the help of the treatment published by Millet et al. [16]. Fig. 6 plots the time dependency of the rate of the surface pressure increase (d˘/dt). Fig. 6a shows the results for gelatin 65B in acetate buffer and in water, and Fig. 6b for gelatin 280B in water and acetate buffer. We observe a first step during which (d˘/dt) changes merely. However, data are quite noisy. For a more clear presentation, the very noisy initial phase was cut for the different curves. More important, subsequent to this phase, all curves
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fall on the same line being linear with 1/t. The slope of (d˘/dt) versus time amounts approximately to −1 in all cases studied, i.e. gelatin 65B and gelatin 280B in acetate buffer or water. Once the air/water interface is covered with gelatin, there is a continuous slowing down of the rate of the surface pressure increase, which is not correlated to the bulk properties. Such logarithmic kinetics is a sign of self-inhibited systems. Millet et al. [16] interpret the behaviour on the basis of a theoretical model developed by Ligoure and Leibler [20] for neutral diblock copolymers from specific solvents. At times larger than the time to reach a critical surface pressure, adsorbed chains form a dense brush creating steric hindrance to further adsorption in the form of dangling chains in the subsurface. According to the model, the diffusion of free chains in the solvent does not control the adsorption kinetics. Once the brush has formed, additional chains have to stretch out in the vicinity of the interface and adopt a reptation-diffusion mechanism in order to penetrate the brush. The authors show that in this case a logarithmic variation of the surface excess amount of molecules ( ) with time is obtained. To compare to the experimental data, a relationship between and ˘ has to be obtained. With the help of the general expression ˘ ∝ n , where n expresses the type of interaction predominant in the adsorption layer, the slope of the curve (d˘/dt) versus time can be computed. It continuously decreases with time, approaching asymptotically −1 in the limit of infinite time. The mathematical treatment is given by Millet et al. [16]. The slopes calculated for the different samples measured here show the value of −1 within the experimental uncertainty. The adsorption of gelatin to the air/water interface can thus reasonably be interpreted within the framework of this theory, although the chemical structure of the gelatin molecule is rather far from a neutral diblock copolymer with a sharp molar weight distribution. However, we suppose that there is the formation of an encumbered surface layer with dangling gelatin chains into the solvent. Additional evidence for the formation of an encumbered interface sub-layer might come from the direct measurement of the thickness of interfacial films. Observation of the surface layer of gelatin by X-ray reflectivity at 40 ◦ C showed that there are two types of dense regions with a layer thickness of 14 Å and an extended region of 94 Å containing only a small percentage of gelatin (5 vol%). This gives a layer thickness of approximately 108 Å, having very high roughness [21]. The surface layers measured with this technique are smaller than the ones from other techniques, because X-ray reflectivity cannot resolve extended chains in the bulk. The surface coverage measured is 1.7 mg/m2 . The surface layer of gelatin on the polystyrene/water interface was measured by small angle neutron scattering (SANS). The layer thickness was approximately 130 Å with a surface coverage of 1.9 mg/m2 [19]. Those layers are still thin compared to the thickness measured in ellipsometry on hydrophobic silica surfaces, amounting to 400 Å (values taken from Ref. [19]). Gelatin is a polyampholyte. Therefore it is capable to develop cationic, anionic and hydrophobic interactions, having binding sites irregularly arranged. This allows the molecules to associate with the interface by electrostatic and/or hydrophobic interactions. We observe with the help of the analysis of the viscosity a large influence of the salt concentration of the solvent on the chain expansion. However, the theory presented here was developed for neutral diblock copolymers. Therefore, we have supposed implicitly that electrostatic interactions can be neglected in our case. Actually, the (d˘/dt) curves regroup to a single curve for one sample, no matter the solvent and the kinetics, and are unaffected by the salt concentration (Fig. 6). The adsorbed layer is probably self-screened due to interactions between the gelatin molecules, so that no repulsing
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ics on the air/water surface were done in the dilute regime. The analysis of the kinetics of the surface pressure increase leads us to the following conclusions:
Fig. 7. Relationship between the equilibrium surface pressure and the gelatin concentration at 42 ◦ C. The lines are drawn to guide the eye. Symbols: gelatin 65B in acetate buffer (), gelatin 65B in water (䊉), gelatin 280B in acetate buffer (), gelatin 280B in water ().
effect between adsorbed layer and arriving molecules is detected, which would cause a delay in adsorption kinetics. Furthermore, we observe no effect of the chains size of gelatin comparing the (d˘/dt) versus time curves for both samples. Comparing the axis of Fig. 6a and b, we see that there is actually no delay between the curves within the experimental uncertainty. One might have expected a delay in the (d˘/dt) versus time curve to longer times for samples with higher molecular size. We think that this is not observed due to the polymolecular character of gelatin, which contains in both samples longer and smaller chains. Fig. 7 plots the results of the equilibrium surface pressure versus gelatin concentration for both samples in both solvents. There is no difference between the equilibrium values for the 280B gelatin in function of the solvent within the experimental uncertainty (±2 mN/m), suggesting that there is no influence of salt concentration on the adsorption and structuring of 280B gelatin on the interface. The equilibrium surface pressure results of the 65B gelatin in water seem to be more stretched on the ordinate axis direction, resulting in low equilibrium surface pressures at low concentrations and higher values at high concentrations. We attribute the behaviour to the polymolecularity of the gelatin samples, containing also branched chains. The sample 65B contains a higher proportion of smaller gelatin chains which might remain more flexible at the interface and thereby allow a higher surface coverage at higher bulk solutions. 4. Conclusion The behaviour of two gelatin samples in solution and at the air/water interface was investigated at 42 ◦ C in order to ensure the coil conformation of the protein molecules. We observe an expansion of gelatin upon dilution in water due the polyelectrolyte nature of the protein chain. The increase of ionic strength of the solvent reduces successfully the expansion. We calculated an apparent value of the intrinsic viscosity of both samples which could be successfully used to plot a master curve of the development of the specific viscosity of both samples on a reduced concentration scale. According to the critical overlap concentration all adsorption kinet-
(1) The analysis of the adsorption kinetics revealed differences between both gelatin samples in terms of the length of the induction phase and the dependency of the equilibrium pressure on the sample concentration. We interpret that there are roughly two populations of gelatin molecules contained in the samples, one having small and the other having long chain length, which are relevant for the adsorption behaviour. The smaller chains adsorb faster to the interface bringing about smaller induction times. Therefore gelatin 65B, being more hydrolyzed, had smaller induction periods. However, at very small gelatin concentrations (10−5 wt%) the sample being composed of mainly larger chains showed the smaller induction time. This effect might be due to a cooperative effect of the long chains, where the adsorption of the subunits of an already attached chain might be easier than the adsorption of a new chain. A second effect of the polymolecular character of the gelatin samples was that the more hydrolyzed sample showed on one hand higher equilibrium surface pressures at high bulk concentrations than the less hydrolyzed sample, but on the other hand smaller equilibrium surface pressures at small bulk concentrations. We think that the same cooperative effect caused this behaviour at small concentrations. (2) The analysis of the adsorption kinetics showed a continuous slow down of the rate of surface pressure increase after the initial adsorption phase, which is linear on a logarithmic time scale with a slope of −1. This behaviour is characteristic for self-inhibited systems. It might be caused by the formation of an encumbered subsurface layer which causes steric hindrance of the newly arriving chains. The adsorption kinetics is analogous to the kinetics of surface-active copolymers, which form a brush on the interface with dangling ends in the solution. Our interpretation might be supported by the observation of surface film layers with high thickness by direct methods, such as ellipsometry or SANS. (3) The adsorption kinetics seems to be independent from the salt concentration of the solvent, although the ionic strength had a high impact on the expansion of the gelatin chains. Once the first, diffusive phase, accomplished, the adsorption kinetics are independent of the gelatin chain length, a behaviour observed also for other surface active polymers.
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