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Chitin nanocrystals for Pickering high internal phase emulsions. Emilie Perrin, Herve Bizot, Bernard Cathala, and Isabelle Capron Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm5010417 • Publication Date (Web): 02 Sep 2014 Downloaded from http://pubs.acs.org on September 6, 2014
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Chitin nanocrystals for Pickering high internal phase emulsions. Emilie Perrin, Hervé Bizot, Bernard Cathala and Isabelle Capron* UR1268 Biopolymères Interactions Assemblages, INRA, F-44316 Nantes, France
ABSTRACT Chitin is a natural polymer of glucosamine bearing N-acetyl groups. Chitin nanocrystals (ChiNC), resulting from the acid hydrolysis of amorphous regions of chitin, are crystalline positively charged rod-like particles. ChiNC show some interfacial properties and very efficiently stabilize oil/water interfaces, leading to the so-called Pickering emulsions. In accordance with the irreversible adsorption of particles, these Pickering emulsions proved stable over time, with constant emulsion volume for several months, even though natural creaming may occur. The emulsions produced are not clearly susceptible to ionic strength or pH in terms of average droplet diameter. However, when mixed with a large amount of oil, high internal phase emulsions (HIPE) containing up to 96% of internal phase are formed as a gel with a texture that can be modulated from soft to solid gel by adjusting concentration, pH and ionic strength.
Keywords: chitin nanocrystal, whiskers, colloidal particle, Pickering emulsion, HIPE, emulsion gel
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INTRODUCTION Renewable natural nanocrystals derived from polysaccharides, such as cellulose, chitin and starch, have attracted great attention owing to their advantages over traditional inorganic or synthetic nanoparticles. These advantages include easy availability, low density, biocompatibility and biodegradability.1 Nevertheless, research focused on chitin nanocrystals (ChiNC) is far less developed than that of other biopolymers such as starch or cellulose, whereas chitin appears to be of strategic importance given its recognized functionalities as an immune modulator and anti-inflammatory agent, and to its role in antithrombogenesis and cell viability.2, 3 In addition, their large surface area and Young's modulus make ChiNC relevant candidates as fillers in polymer nanocomposites.4-6 Furthermore, dispersed in aqueous media, ChiNC forms stable colloidal suspensions and, at higher concentrations, liquid crystalline phases occur and align cooperatively, leading to cholesteric order.7-9 Chitin is synthesized in a large number of living organisms and, considering the amount of chitin produced annually on the global scale, it is considered the second most abundant polysaccharide after cellulose.10 Chitin occurs in nature as ordered crystalline microfibrils that act as structural components in the exoskeleton of arthropods and in the cell walls of fungi and yeast. Similarly to cellulose whiskers, some crystalline particles can be isolated by acid hydrolysis. Since the first preparation of chitin whiskers by Marchessault et al.,11 a variety of sources has been identified for the preparation of chitin whiskers, including crab shells,9, 12, 13 shrimp shells,14 squid pens,15 the tubes of Tenvia jerichonana16 and Riftia pachyptila tube worms,17 where chitin is generally associated with proteins, pigments and calcium carbonate.18
Chemically, chitin is a linear polymer of β (1–4) linked acetyl-D-glucosamine residues where acetamide groups are positioned at the C2 position of the D-glucopyranose unit. These
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residues make it slightly hydrophobic. However, it occurs naturally partially deacetylated at the fiber level depending on the source.10 As a result, glucosamine units with primary amino groups are always present on the chitin crystallite surface and are protonated in acid media. The ratio of glucosamine residues to total residues is referred to as the degree of deacetylation (DD) or similarly to the ratio of acetyl-glucosamine residues to total residues referred to as the degree of acetylation (DA). Deacetylation over approximately 50% of the N-acetyl groups leads to disruption of the crystalline structure via electrostatic repulsions and results in chitosan, a water-soluble polymer, which is the most common chitin derivative in terms of applications. It has already been reported that colloidal particles are able to stabilize oil/waterinterfaces, forming the so-called highly stable Pickering emulsions.19-21 Chitin nanocrystals can form such oil-in-water emulsions either by synergy with surfactants,22 or as unique stabilizer23 similarly to those described with cellulose.24 Tzoumaki et al. proposed that ChiNC adsorb at the O/W emulsions, forming a gel-like microstructure composed of flocculated oil droplets.23 As particular emulsions, high internal phase emulsions (HIPE) are defined as emulsions with an internal phase volume fraction (φ) of 0.74 or greater.25-29 HIPE are used in numerous applications, including food preparation, oil recovery, cosmetics and, recently, as templates in material science for thermal insulation, buoyancy, and packaging.27, 29, 30 In the present work, we have studied the various parameters involved in the stabilization of ChiNC at the oil/water interface of individual droplets forming stable Pickering emulsions. Furthermore, we report, for the first time, the stabilization by ChiNC of Pickering-HIPE as a gel including up to 96% of oil. It is also demonstrated how the gel structuration can be modulated using the preparation parameters.
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EXPERIMENTAL SECTION Materials: Chitin powder was obtained from shrimp shell (France Chitine). Styrene (Fluka) was purified
by percolation
through
activated
alumina
(EcoChrom
N super1).
Azobisisobutyronitrile (abbreviated AIBN) as the free-radical polymerization thermoinitiator was from Sigma-Aldrich. Cyclohexane and hexadecane was purified by extensive extraction with water to remove most of the contaminating surfactants. All of the reagents used were of analytical grade (Sigma-Aldrich), and water was purified with the Milli-Q reagent system (Millipore).
Preparation of ChiNC: ChiNC were prepared from the hydrolysis of chitin powder with 3N HCl without deproteinization in KOH or NaOH. Only a swelling pre-process with HCl followed by centrifugation was carried out in order to wash the powder. After that, ChiNC were prepared mostly following the procedure proposed by Revol et al.7 Four grams of chitin were submitted to acid hydrolysis in 80 mL of boiling 3N HCl for 90 min. The resulting slurry was washed with Milli-Q water by successive centrifugations for 20 min at 10000 g, resuspended in water and dialyzed against 0.01 mM HCl for 4 days. The suspension was sonicated (QSonica Sonicator, 700 w) in order to disperse the remaining aggregates with intermittent cycles for a total operating time of 10 min. The suspension was then centrifuged three times from 10000 to 15000 g to remove the residual debris, and filtrated through 5-µm and 1.2-µm pore size cellulose nitrate membranes (Millipore). The suspension was stored at 4°C.
Transmission electron microscopy (TEM): A total of 20 µL of a ChiNC suspension in 0.01 mM HCl (0.0025% w/v) was deposited on a freshly glow-discharged carbon-coated electron microscope grid (200 mesh copper; Electron Microscopy Sciences, USA) and the excess
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removed by absorption on a filter paper. Negative staining was performed by adding 20 µL of uranyl acetate solution (2% w/v). The excess solution was removed and the grids were dried at room temperature. The grid was observed under standard conditions with a JEOL JEM1230 transmission electron microscope operating at 80 kV. The image analysis using image J software was performed on 280 nanocrystal measurements.
Degree of deacetylation (DD): Conductometric titration was carried out on never-dried ChiNC suspensions at 3.5 g/L in 2 mM HCl to ensure complete protonation of the amino groups. The suspensions were stirred under nitrogen for 10 min. Conductometric titration was then performed with freshly prepared 10 mM NaOH degassed under nitrogen with a TIM900 titration manager and a CDM230 conductimeter equipped with a CDC749 titration cell (Radiometer, Denmark). More details for DD determinations can be found in the SI-1.
Preparation of the ChiNC stabilized emulsions: The oil-in-water (O/W) Pickering emulsions were prepared using hexadecane and a ChiNC aqueous suspension at the required concentrations of ChiNC, salt and HCl. All the emulsions were prepared using an oil/aqueous phase ratio of 20/80 and kept above the melting temperature of hexadecane. The preparation was sonicated using intermittent pulses. Droplet diameter was measured by laser light diffraction using a Mastersizer 2000 granulometer apparatus equipped with a He_Ne laser (Malvern Instruments, U.K.). Two protocols were used to prepare a high internal phase emulsion: (1) a direct emulsion for which the required volumes of aqueous suspension and hexadecane were mixed using a rotorstator (double-cylinder-type homogenizer) for 30s to 2 min according to the emulsion volume at 6000 rpm; (2) a two-step protocol which involved a Pickering pre-emulsion was prepared
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according to the protocol above and mixed to the added oil using a rotor-stator for 30 s to 2 min according to the emulsion volume at 6000 rpm.
Scanning electron microscopy (SEM): In order to visualize the droplets, some styrene-inwater emulsions were prepared and polymerized. Hexadecane and styrene have similar surface tension (27 mN/m and 32 mN/m, respectively), and we have controlled by optical microscopy that they similarly led to stable emulsions with the same average droplet diameters. Therefore, 0.15 mL of a styrene/AIBN mixture (ratio 100:1 w/w) was mixed with 1.35 ml of ChiNC dispersed at 3 g/L in 20 mM NaCl at pH5, and degassed with nitrogen gas for 10 min. The emulsion was made via sonication, degassed again with nitrogen gas for 10 min and polymerized at 50°C overnight. The resulting beads were washed by repeated centrifugations. Dried beads were metalized with platinum and visualized with a JEOL 7600F instrument (on electron microscopy platform IMN, Nantes, France). For foam visualization, the emulsions were prepared using cyclohexane instead of hexadecane as described in Tasset et al.,31 freeze-dried and subsequently metalized with platinum.
RESULTS AND DISCUSSION Basic characterizations of ChiNC - Transmission electron microscopy (TEM) as shown on Figure 1a reveals elongated nanoparticles. In several cases, fragments were observed as well as associations. The size distribution has typical histograms, as shown in Figure 1, with an average of 160 ± 77 nm in length and 16 ± 5 nm in width. After drying a thin film of ChiNC suspension on a silicon wafer, a thickness of 10 nm was measured by ellipsometry.32
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100
a)
120
b)
80
c)
100 80
60
Count
Count
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40
60 40
20
20 0 0
100
200 300 Length (nm)
400
0 0
10
20 30 Width (nm)
40
Figure 1: TEM image (a) and size distribution in length (b) and width (c)
The surface charge density was evaluated by conductometric titration to quantify charged groups of nanocrystals.9, 33-36 The samples were prepared at pH 3,34 and titrated using NaOH, revealing three-slope curves (see SI for details). The result obtained was 0.48 ± 0.02 charge/nm².
In
surface
amino
groups,
this
corresponds
to
approximately 4000
charges/particle. Taking into account that an ideally fully deacetylated sample would bear about 1.7 charges/nm², about 28% of the acetylated groups are modified into amine functions.
Interfacial properties of ChiNC and preparation of Pickering emulsions – ChiNC showed interfacial properties similar to those demonstrated for cellulose nanocrystals (CNC),24,
36, 37
in particular, the ability to stabilize oil-in-water Pickering emulsions.23
However, and contrary to CNC, chitin is susceptible to both ionic strength and pH variations due to the amino groups. The amino group on chitin has a pKa of 6.1. This means that at a pH of approximately 6, half of the NH2 groups present are uncharged and half are in cationic form. At pH 3, it is considered that all the amines are positively charged.8 On the other hand, it precipitates at pH values above 6.5 and will react to ionic strength. Different parameters have been tested in order to take the electrostatic interactions on the emulsion formation into account.
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Effect of ionic strength and pH on the emulsions process - The two main parameters involving the flocculating, ionic strength and pH, have been studied. Figure 2 gives a global view of their effect on emulsification. It clearly appears from the emulsion prepared at pH 5 (0.01 mM HCl) in the absence of salt, that a small fraction of the ChiNC stabilized a few droplets (low emulsion volume), but the major part of the ChiNC is in suspension in the aqueous lower phase, and a bimodal droplet diameter distribution was obtained (Figure 3a). As already observed for charged cellulose36, unscreened repulsive interactions lead to an incomplete coverage of the surface of the droplets and consequently to partial coalescence. In contrast, as soon as electrolyte is added, even with only 2 mM NaCl, the complete oil volume is stabilized in emulsion form, and the creaming process, due to the low density of the oil phase, shows a transparent aqueous phase. This reveals the necessity to screen the charges in order to hinder the repulsions and allow densification at the interface. Furthermore, the creaming process is generally identified as a destabilizing process. No variation in emulsion volume was observed here over a period of several months showing that the creaming process occurs without coalescence.
Figure 2: Illustration of the different emulsions prepared with the various concentrations given in the table
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In all the conditions tested, except when no ionic strength was added, monomodal populations of very similar diameter were obtained regardless of the ionic strength and pH as illustrated by the superimposition of the distribution curves (Figures 3). As a result, as long as an ionic force of 2 mM is added, and regardless of the pH, emulsions with very homogeneous size distributions are obtained.
Figure 3: Average droplet diameter variation with a concentration of 3 g/L in the aqueous phase using a 20/80 oil/water ratio a) with different NaCl concentrations in 0.01 mM HCl and b) with different HCl concentrations in 20 mM NaCl
To evaluate the stability, centrifugation accelerates the creaming process, forcing the droplets to concentrate.24 The excess water is then excluded, leading to close packing conditions. Centrifugation at 10000 g was performed on emulsions prepared at 3g/L with 20 mM NaCl and pH 5 and the same monomodal droplet size distribution as before centrifugation was maintained. In addition, samples were stored for one week at 4°C, 20°C, and 40°C, in sealed tubes in order to monitor their stability. The droplet size distribution showed no variation,
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revealing the very high stability of these ChiNC-stabilized emulsions (Figure in SI-2) generally associated to colloids stabilized emulsions.
Effect of concentration of ChiNC on the drop size – When the concentration of ChiNC is increased, as demonstrated in Figure 2 for an aqueous phase at 5 g/L compared to 3 g/L, the emulsion volume increases up to fill the complete volume. The contribution of the droplet size was monitored by measuring the surface mean diameter D(3,2) while varying concentrations of ChiNC in the aqueous phase for a fixed amount of oil. Results are given for a ratio of 20/80 (O/W) (Figure 4). Stable droplets with larger sizes were measured at the lowest concentrations and the size decreased to a diameter of 4.5 µm at 5 g/L of ChiNC in the aqueous phase. This coalescence previously reported for other Pickering emulsions38,39 is based on a limited coalescence process.40,41 This occurs when sonication produces small droplets, i.e. larger areas of oil–water interface than can potentially be covered by the nanocrystals. When sonication is stopped, the partially unprotected droplets coalesce until the interface is sufficiently covered. The droplet diameter can then be modulated with the weight of ChiNC (Figure 4). This is in agreement with the irreversible adsorption of the ChiNC at the oil / water interface. At higher concentrations, this diameter reaches a plateau value at which a denser layer is accompanied by an increased stability of the droplets.
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Figure 4: Average diameter (µm) vs. ChiNC concentration in the aqueous phase (g/L)
As a result, ChiNC proved to bend at the oil/water interface, as illustrated by the SEM images on Figure 5. The coverage of the beads is defined as the ratio of the particles area available to stabilize the interface, and the calculated total area of the oil droplets taking the D(3,2) droplet diameter measured for each emulsion into account.24,
42
A value of approximately 100%
coverage was calculated, revealing the high coverage ability of these solid particles. As can be seen in Figure 5, some crystals overlap, leading to a rigid armored steric protection that inhibits coalescence responsible for their exceptional stability.
1 µm
250 nm
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Figure 5: SEM images of polymerized styrene/water emulsions stabilized by chitin nanocrystals
Pure ChiNC Pickering high internal phase emulsion with modulated cell diameter – High internal phase emulsions (HIPE) are emulsions with an internal phase volume (φ) above 74%.25, 26 The droplets in these highly concentrated emulsions can possibly share interfacial particles within a continuous scaffold,28 provided the particles can be bound or locked together in a continuous structure. Adding hexadecane to the initial emulsion under stirring with a double-cylinder-type homogenizer, resulted in a new gel-like structure with internal phase volume ratios up to 96% (Figure 6). There are generally two ways to reach such a high percentage: (i) increase the polydispersity of the droplet diameters, filling the holes between the droplets; and (ii) deforming the droplets in order to decrease the empty inter-droplet space. In the case of ChiNC, both conditions contribute to the high internal phase ratio. This surprisingly stable nanometric structuration where deformation of the droplets appears without coalescence was already observed with CNC.28 However, using CNC a two-step process was required, including a first emulsion preparation followed by a second step during which oil was added. In contrast, ChiNC may produce similar highly stable gel structure directly from the initial ChiNC suspension. This suggests that less energy was required to stack the nanocrystals at the interface. This could be attributed to the less hydrophilic N-acetyl group in C2 position of the anhydroglucose where cellulose has a hydroxyl function. The same maximum internal phase volume fraction was obtained using the direct or indirect method (Figure 6) but a slight difference was observed between 50% and 75% of internal phase and a stronger deformable visco-elastic gel was formed when using the two-step 12 ACS Paragon Plus Environment
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process. This is attributed to the sonication used in the two-step process. Ultrasonication, providing a high energy, implies a better dispersion of the ChiNC in suspension when the emulsion is prepared. This might lead to a higher surface coverage with more individualized ChiNC and may allow a better structuration of the interfaces.
Figure 6: Percentage of internal phase obtained for ChiNC at 3 g/L in the aqueous phase with 20 mM NaCl and 0.01 mM HCl using a two-step process for which a first emulsion is prepared before introducing the rest of the oil and a direct process for which the oil is directly included to the suspension of ChiNC.
The resulting emulsions prepared with a two-step method are shown Figure 7 according to both the ChiNC concentration and the NaCl concentration, poured directly from the tube onto glass cups. These two parameters revealed the same tendency to increase the structuration of the gel. At low concentrations and low ionic strengths, a soft gel is obtained that can flow on a surface but remain cohesive. Increasing either ChiNC concentration from 1.5 g/L to 3 g/L, or salt concentration from 50 to 100 mM, the gel no longer spreads out on the glass but 13 ACS Paragon Plus Environment
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maintains its initial form. The gel can then be cut into slices that have a solid visco-elastic gel texture.
1.5 g/L
3 g/L
100 mM
50 mM
Figure 7: Emulsion gels formed in various conditions of ChiNC concentration and ionic strength.
The initial droplet sizes were identical as demonstrated figure 3. Also the same percentage of oil was stabilized for each emulsion gel whatever the ionic strength conditions (Fig. SI-3). However, figure 7 shows that the addition of salt strengthens the walls, forming a denser texture. The gel strength of the emulsion gel can then be easily monitored according to ionic strength and ChiNC concentration (Fig. SI-4).
Since the ChiNC are irreversibly adsorbed at the interface, this gel formation is explained by variation of the droplet diameters. When oil is added there is no particle desorption but a swelling of the droplets. Concentrated emulsions can thereby be formed without coalescence leading to a gel. In order to visualize it, emulsions were prepared using cyclohexane, freezedried and metalized with platinum for SEM visualization.
31
The internal structuration
variation with the added oil phase was monitored up to 64% of internal phase, revealing the
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swelling of the droplets. This procedure is similar to those observed. The initial droplets with a diameter of less than 10 µm, as described in the first part of this work, disappeared in favor of a population of larger-size droplets of several tenths of a micron in diameter (Figure 8). The starting concentration in ChiNC in the aqueous phase was 3 g/L, which corresponds to 2.4 g/L considering the whole emulsion (80% of aqueous phase / 20% of internal phase). In the structures presented on the SEM images where additional oil is included, it reaches 36%, 54% and 64% of internal phase, corresponding to amounts of ChiNC of 1.9 g/L, 1.4 g/L and 1.0 g/L of emulsion, respectively. This means that such a structured gel or cellular foam can even be obtained at 0.1% of ChiNC without any additional surface-active material. Furthermore, it demonstrates the strength of such internal structuration since no collapse occurred while drying.
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Figure 8: SEM images of solid emulsions from freeze-dried cyclohexane-in-water emulsions stabilized by ChiNC, to which cyclohexane is added up to the percentage of internal phase indicated. Three different magnifications for each sample are shown (x 100, x 400, and x 1000 or x 2000).
CONCLUSIONS It appears that ChiNC can be used as colloidal particles capable of very efficiently stabilizing interfacial areas and produce highly stable oil-in-water Pickering emulsions. Furthermore, their high stability makes it possible to introduce a large part of internal phase to produce high internal phase emulsion (HIPE) up to a percentage of hydrophobic internal phase as high as 96%. This HIPE produces a gel whose texturing depends on the ionic strength, pH and concentration. It is also easy to handle since, compared to what was observed with cellulose, such emulsions might be produced according to a one-step process where the constituents are mixed directly. As a result, these emulsions that use unmodified ChiNC proved to be in a same time very stable and versatile enough to be modulated from liquid to gel texture. Furthermore, the emulsion is also able to form stable solid foams when both liquid phases are removed using the freeze-drying process without destructuration of the walls of the emulsion. These cellular foams show that the cell dimensions might vary with the oil added to the system.
AKNOWLEDGMENTS The authors would like to thank Joelle Davy for SEM imaging and Nicolas Stephan for SEM assistance (IMN, Nantes, France), Brigitte Bouchet for her assistance in TEM experiments
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(BIBS Platform, INRA Nantes, France) and the Pays de la Loire Regional Council (Matières Network) for financial support.
SUPPORTING INFORMATION Data for surface charge density determination, stability to temperature, effect of ionic strength on the percentage of internal phase and the comparison of the internal phase ratio according to different parameters for HIPE formation is available in SI document. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author * E-mail address: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.
REFERENCES 1. Klemm, D.; Kramer, F.; Moritz, S.; Lindstrom, T.; Ankerfors, M.; Gray, D.; Dorris, A., Angew. Chem. Int. Ed. 2011, 50, 5438-5466. 2. Lee, C. G.; Da Silva, C. A.; Dela Cruz, C. S.; Ahangari, F.; Ma, B.; Kang, M. J.; He, C. H.; Takyar, S.; Elias, J. A., Role of Chitin and Chitinase/Chitinase-Like Proteins in Inflammation, Tissue Remodeling, and Injury. In Annual Review of Physiology, Julius, D.; Clapham, D. E., Eds. 2011; Vol. 73, pp 479-501.
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3. Muzzarelli, R. A. A.; Boudrant, J.; Meyer, D.; Manno, N.; DeMarchis, M.; Paoletti, M. G., Carbohydr. Polym. 2012, 87, 995-1012. 4. Lin, N.; Huang, J.; Dufresne, A., Nanoscale 2012, 4, 3274-3294. 5. Habibi, Y.; Dufresne, A., Biomacromolecules 2008, 9, 1974-1980. 6. Zeng, J.-B.; He, Y.-S.; Li, S.-L.; Wang, Y.-Z., Biomacromolecules 2012, 13, 1-11. 7. Revol, J. F.; Marchessault, R. H., Int. J. Biol. Macromol. 1993, 15, 329-335. 8. Li, J.; Revol, J. F.; Marchessault, R. H., J. Appl. Polym. Sci. 1997, 65, 373-380. 9. Belamie, E.; Davidson, P.; Giraud-Guille, M. M., J. Phys. Chem. B 2004, 108, 14991-15000. 10. Rinaudo, M., Prog. Polym. Sci. 2006, 31, 603-632. 11. Marchessault, R. H.; Morehead, F. F.; Walter, N. M., Nature 1959, 184, 632-633. 12. Nair, K. G.; Dufresne, A., Biomacromolecules 2003, 4, 657-665. 13. Tzoumaki, M. V.; Moschakis, T.; Biliaderis, C. G., Biomacromolecules 2010, 11, 175-181. 14. Sriupayo, J.; Supaphol, P.; Blackwell, J.; Rujiravanit, R., Polymer 2005, 46, 5637-5644. 15. Paillet, M.; Dufresne, A., Macromolecules 2001, 34, 6527-6530. 16. Saito, Y.; Putaux, J. L.; Okano, T.; Gaill, F.; Chanzy, H., Macromolecules 1997, 30, 3867-3873. 17. Morin, A.; Dufresne, A., Macromolecules 2002, 35, 2190-2199. 18. Percot, A.; Viton, C.; Domard, A., Biomacromolecules 2003, 4, 12-18. 19. Ramsden, W., Proc. Roy. Soc. 1903, 72, 156. 20. Pickering, S. U., J. Chem. Soc. 1907, 91, 2001. 21. Binks, B. P., Curr. Opin. Colloid Interface Sci. 2002, 7, 21-41. 22. Magdassi, S.; Neiroukh, Z., J. Dispersion Sci. Technol. 1990, 11, 69-74. 23. Tzoumaki, M. V.; Moschakis, T.; Kiosseoglou, V.; Biliaderis, C. G., Food Hydrocolloids 2011, 25, 1521-1529. 24. Kalashnikova, I.; Bizot, H.; Cathala, B.; Capron, I., Langmuir 2011, 27, 7471-7479. 25. Lissant, K. J.; Mayhan, K. G. J., J. Colloid Interface Sci. 1973, 42, 201-208. 26. Cameron, N. R.; D.C., S., Adv. Polym. Sci. 1996, 126, 163-214. 27. Brun, N.; Ungureanu, S.; Deleuze, H.; Backov, R., Chem. Soc. Rev. 2011, 40, 771-788. 28. Capron, I.; Cathala, B., Biomacromolecules 2013, 14, 291-296. 29. Cameron, N. R., Polymer 2005, 46, 1439-1449. 30. Binks, B. P., Adv. Mater. 2002, 14, 1824-1827. 31. Tasset, S.; Cathala, B.; Bizot, H.; Capron, I., Rsc Advances 2014, 4, 893-898. 32. Villares, A.; Moreau, C.; Capron, I.; Cathala, B., Biomacromolecules 2014, 15, 188-194. 33. Raymond, L.; Morin, F. G.; Marchessault, R. H., Carbohydr. Res. 1993, 246, 331. 34. Li, J.; Revol, J. F.; Naranjo, E.; Marchessault, R. H., Int. J. Biol. Macromol. 1996, 18, 177-187. 35. Gousse, C.; Chanzy, H.; Cerrada, M. L.; Fleury, E., Polymer 2004, 45, 1569-1575. 36. Kalashnikova, I.; Bizot, H.; Cathala, B.; Capron, I., Biomacromolecules 2012, 13, 267-275. 37. Kalashnikova, I.; Bizot, H.; Bertoncini, P.; Cathala, B.; Capron, I., Soft Matter 2013, 9, 952-959. 38. Tambe, E. T.; Sharma, M. M., J. Colloid Interface Sci. 1993, 157, 244-253. 39. Aveyard, R.; Binks, B. P.; Clint, J. H., Adv. Colloid Interface Sci. 2003, 100, 503-546. 40. Whitesides, T. H.; Ross, D. S., J. Colloid Interface Sci. 1995, 169, 48-59. 41. Arditty, S.; Whitby, C. P.; Binks, B. P.; Schmitt, V.; Leal-Calderon, F., Eur. Phys. J. E 2003, 11, 273-281. 42. Gautier, F.; Destribats, M.; Perrier-Cornet, R.; Dechezelles, J. F.; Giermanska, J.; Heroguez, V.; Ravaine, S.; Leal-Calderon, F.; Schmitt, V., Phys. Chem. Chem. Phys. 2007, 9, 6455-6462.
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Table of Contents Graphic
Emulsion gel
Liquid emulsion
Oil > 90%
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Chitin nanocrystals for Pickering high internal phase emulsions. Emilie Perrin, Herve Bizot, Bernard Cathala, and Isabelle Capron Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm5010417 • Publication Date (Web): 02 Sep 2014 Downloaded from http://pubs.acs.org on September 6, 2014
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Chitin nanocrystals for Pickering high internal phase emulsions. Emilie Perrin, Hervé Bizot, Bernard Cathala and Isabelle Capron* UR1268 Biopolymères Interactions Assemblages, INRA, F-44316 Nantes, France
ABSTRACT Chitin is a natural polymer of glucosamine bearing N-acetyl groups. Chitin nanocrystals (ChiNC), resulting from the acid hydrolysis of amorphous regions of chitin, are crystalline positively charged rod-like particles. ChiNC show some interfacial properties and very efficiently stabilize oil/water interfaces, leading to the so-called Pickering emulsions. In accordance with the irreversible adsorption of particles, these Pickering emulsions proved stable over time, with constant emulsion volume for several months, even though natural creaming may occur. The emulsions produced are not clearly susceptible to ionic strength or pH in terms of average droplet diameter. However, when mixed with a large amount of oil, high internal phase emulsions (HIPE) containing up to 96% of internal phase are formed as a gel with a texture that can be modulated from soft to solid gel by adjusting concentration, pH and ionic strength.
Keywords: chitin nanocrystal, whiskers, colloidal particle, Pickering emulsion, HIPE, emulsion gel
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INTRODUCTION Renewable natural nanocrystals derived from polysaccharides, such as cellulose, chitin and starch, have attracted great attention owing to their advantages over traditional inorganic or synthetic nanoparticles. These advantages include easy availability, low density, biocompatibility and biodegradability.1 Nevertheless, research focused on chitin nanocrystals (ChiNC) is far less developed than that of other biopolymers such as starch or cellulose, whereas chitin appears to be of strategic importance given its recognized functionalities as an immune modulator and anti-inflammatory agent, and to its role in antithrombogenesis and cell viability.2, 3 In addition, their large surface area and Young's modulus make ChiNC relevant candidates as fillers in polymer nanocomposites.4-6 Furthermore, dispersed in aqueous media, ChiNC forms stable colloidal suspensions and, at higher concentrations, liquid crystalline phases occur and align cooperatively, leading to cholesteric order.7-9 Chitin is synthesized in a large number of living organisms and, considering the amount of chitin produced annually on the global scale, it is considered the second most abundant polysaccharide after cellulose.10 Chitin occurs in nature as ordered crystalline microfibrils that act as structural components in the exoskeleton of arthropods and in the cell walls of fungi and yeast. Similarly to cellulose whiskers, some crystalline particles can be isolated by acid hydrolysis. Since the first preparation of chitin whiskers by Marchessault et al.,11 a variety of sources has been identified for the preparation of chitin whiskers, including crab shells,9, 12, 13 shrimp shells,14 squid pens,15 the tubes of Tenvia jerichonana16 and Riftia pachyptila tube worms,17 where chitin is generally associated with proteins, pigments and calcium carbonate.18
Chemically, chitin is a linear polymer of β (1–4) linked acetyl-D-glucosamine residues where acetamide groups are positioned at the C2 position of the D-glucopyranose unit. These
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residues make it slightly hydrophobic. However, it occurs naturally partially deacetylated at the fiber level depending on the source.10 As a result, glucosamine units with primary amino groups are always present on the chitin crystallite surface and are protonated in acid media. The ratio of glucosamine residues to total residues is referred to as the degree of deacetylation (DD) or similarly to the ratio of acetyl-glucosamine residues to total residues referred to as the degree of acetylation (DA). Deacetylation over approximately 50% of the N-acetyl groups leads to disruption of the crystalline structure via electrostatic repulsions and results in chitosan, a water-soluble polymer, which is the most common chitin derivative in terms of applications. It has already been reported that colloidal particles are able to stabilize oil/waterinterfaces, forming the so-called highly stable Pickering emulsions.19-21 Chitin nanocrystals can form such oil-in-water emulsions either by synergy with surfactants,22 or as unique stabilizer23 similarly to those described with cellulose.24 Tzoumaki et al. proposed that ChiNC adsorb at the O/W emulsions, forming a gel-like microstructure composed of flocculated oil droplets.23 As particular emulsions, high internal phase emulsions (HIPE) are defined as emulsions with an internal phase volume fraction (φ) of 0.74 or greater.25-29 HIPE are used in numerous applications, including food preparation, oil recovery, cosmetics and, recently, as templates in material science for thermal insulation, buoyancy, and packaging.27, 29, 30 In the present work, we have studied the various parameters involved in the stabilization of ChiNC at the oil/water interface of individual droplets forming stable Pickering emulsions. Furthermore, we report, for the first time, the stabilization by ChiNC of Pickering-HIPE as a gel including up to 96% of oil. It is also demonstrated how the gel structuration can be modulated using the preparation parameters.
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EXPERIMENTAL SECTION Materials: Chitin powder was obtained from shrimp shell (France Chitine). Styrene (Fluka) was purified
by percolation
through
activated
alumina
(EcoChrom
N super1).
Azobisisobutyronitrile (abbreviated AIBN) as the free-radical polymerization thermoinitiator was from Sigma-Aldrich. Cyclohexane and hexadecane was purified by extensive extraction with water to remove most of the contaminating surfactants. All of the reagents used were of analytical grade (Sigma-Aldrich), and water was purified with the Milli-Q reagent system (Millipore).
Preparation of ChiNC: ChiNC were prepared from the hydrolysis of chitin powder with 3N HCl without deproteinization in KOH or NaOH. Only a swelling pre-process with HCl followed by centrifugation was carried out in order to wash the powder. After that, ChiNC were prepared mostly following the procedure proposed by Revol et al.7 Four grams of chitin were submitted to acid hydrolysis in 80 mL of boiling 3N HCl for 90 min. The resulting slurry was washed with Milli-Q water by successive centrifugations for 20 min at 10000 g, resuspended in water and dialyzed against 0.01 mM HCl for 4 days. The suspension was sonicated (QSonica Sonicator, 700 w) in order to disperse the remaining aggregates with intermittent cycles for a total operating time of 10 min. The suspension was then centrifuged three times from 10000 to 15000 g to remove the residual debris, and filtrated through 5-µm and 1.2-µm pore size cellulose nitrate membranes (Millipore). The suspension was stored at 4°C.
Transmission electron microscopy (TEM): A total of 20 µL of a ChiNC suspension in 0.01 mM HCl (0.0025% w/v) was deposited on a freshly glow-discharged carbon-coated electron microscope grid (200 mesh copper; Electron Microscopy Sciences, USA) and the excess
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removed by absorption on a filter paper. Negative staining was performed by adding 20 µL of uranyl acetate solution (2% w/v). The excess solution was removed and the grids were dried at room temperature. The grid was observed under standard conditions with a JEOL JEM1230 transmission electron microscope operating at 80 kV. The image analysis using image J software was performed on 280 nanocrystal measurements.
Degree of deacetylation (DD): Conductometric titration was carried out on never-dried ChiNC suspensions at 3.5 g/L in 2 mM HCl to ensure complete protonation of the amino groups. The suspensions were stirred under nitrogen for 10 min. Conductometric titration was then performed with freshly prepared 10 mM NaOH degassed under nitrogen with a TIM900 titration manager and a CDM230 conductimeter equipped with a CDC749 titration cell (Radiometer, Denmark). More details for DD determinations can be found in the SI-1.
Preparation of the ChiNC stabilized emulsions: The oil-in-water (O/W) Pickering emulsions were prepared using hexadecane and a ChiNC aqueous suspension at the required concentrations of ChiNC, salt and HCl. All the emulsions were prepared using an oil/aqueous phase ratio of 20/80 and kept above the melting temperature of hexadecane. The preparation was sonicated using intermittent pulses. Droplet diameter was measured by laser light diffraction using a Mastersizer 2000 granulometer apparatus equipped with a He_Ne laser (Malvern Instruments, U.K.). Two protocols were used to prepare a high internal phase emulsion: (1) a direct emulsion for which the required volumes of aqueous suspension and hexadecane were mixed using a rotorstator (double-cylinder-type homogenizer) for 30s to 2 min according to the emulsion volume at 6000 rpm; (2) a two-step protocol which involved a Pickering pre-emulsion was prepared
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according to the protocol above and mixed to the added oil using a rotor-stator for 30 s to 2 min according to the emulsion volume at 6000 rpm.
Scanning electron microscopy (SEM): In order to visualize the droplets, some styrene-inwater emulsions were prepared and polymerized. Hexadecane and styrene have similar surface tension (27 mN/m and 32 mN/m, respectively), and we have controlled by optical microscopy that they similarly led to stable emulsions with the same average droplet diameters. Therefore, 0.15 mL of a styrene/AIBN mixture (ratio 100:1 w/w) was mixed with 1.35 ml of ChiNC dispersed at 3 g/L in 20 mM NaCl at pH5, and degassed with nitrogen gas for 10 min. The emulsion was made via sonication, degassed again with nitrogen gas for 10 min and polymerized at 50°C overnight. The resulting beads were washed by repeated centrifugations. Dried beads were metalized with platinum and visualized with a JEOL 7600F instrument (on electron microscopy platform IMN, Nantes, France). For foam visualization, the emulsions were prepared using cyclohexane instead of hexadecane as described in Tasset et al.,31 freeze-dried and subsequently metalized with platinum.
RESULTS AND DISCUSSION Basic characterizations of ChiNC - Transmission electron microscopy (TEM) as shown on Figure 1a reveals elongated nanoparticles. In several cases, fragments were observed as well as associations. The size distribution has typical histograms, as shown in Figure 1, with an average of 160 ± 77 nm in length and 16 ± 5 nm in width. After drying a thin film of ChiNC suspension on a silicon wafer, a thickness of 10 nm was measured by ellipsometry.32
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100
a)
120
b)
80
c)
100 80
60
Count
Count
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40
60 40
20
20 0 0
100
200 300 Length (nm)
400
0 0
10
20 30 Width (nm)
40
Figure 1: TEM image (a) and size distribution in length (b) and width (c)
The surface charge density was evaluated by conductometric titration to quantify charged groups of nanocrystals.9, 33-36 The samples were prepared at pH 3,34 and titrated using NaOH, revealing three-slope curves (see SI for details). The result obtained was 0.48 ± 0.02 charge/nm².
In
surface
amino
groups,
this
corresponds
to
approximately 4000
charges/particle. Taking into account that an ideally fully deacetylated sample would bear about 1.7 charges/nm², about 28% of the acetylated groups are modified into amine functions.
Interfacial properties of ChiNC and preparation of Pickering emulsions – ChiNC showed interfacial properties similar to those demonstrated for cellulose nanocrystals (CNC),24,
36, 37
in particular, the ability to stabilize oil-in-water Pickering emulsions.23
However, and contrary to CNC, chitin is susceptible to both ionic strength and pH variations due to the amino groups. The amino group on chitin has a pKa of 6.1. This means that at a pH of approximately 6, half of the NH2 groups present are uncharged and half are in cationic form. At pH 3, it is considered that all the amines are positively charged.8 On the other hand, it precipitates at pH values above 6.5 and will react to ionic strength. Different parameters have been tested in order to take the electrostatic interactions on the emulsion formation into account.
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Effect of ionic strength and pH on the emulsions process - The two main parameters involving the flocculating, ionic strength and pH, have been studied. Figure 2 gives a global view of their effect on emulsification. It clearly appears from the emulsion prepared at pH 5 (0.01 mM HCl) in the absence of salt, that a small fraction of the ChiNC stabilized a few droplets (low emulsion volume), but the major part of the ChiNC is in suspension in the aqueous lower phase, and a bimodal droplet diameter distribution was obtained (Figure 3a). As already observed for charged cellulose36, unscreened repulsive interactions lead to an incomplete coverage of the surface of the droplets and consequently to partial coalescence. In contrast, as soon as electrolyte is added, even with only 2 mM NaCl, the complete oil volume is stabilized in emulsion form, and the creaming process, due to the low density of the oil phase, shows a transparent aqueous phase. This reveals the necessity to screen the charges in order to hinder the repulsions and allow densification at the interface. Furthermore, the creaming process is generally identified as a destabilizing process. No variation in emulsion volume was observed here over a period of several months showing that the creaming process occurs without coalescence.
Figure 2: Illustration of the different emulsions prepared with the various concentrations given in the table
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In all the conditions tested, except when no ionic strength was added, monomodal populations of very similar diameter were obtained regardless of the ionic strength and pH as illustrated by the superimposition of the distribution curves (Figures 3). As a result, as long as an ionic force of 2 mM is added, and regardless of the pH, emulsions with very homogeneous size distributions are obtained.
Figure 3: Average droplet diameter variation with a concentration of 3 g/L in the aqueous phase using a 20/80 oil/water ratio a) with different NaCl concentrations in 0.01 mM HCl and b) with different HCl concentrations in 20 mM NaCl
To evaluate the stability, centrifugation accelerates the creaming process, forcing the droplets to concentrate.24 The excess water is then excluded, leading to close packing conditions. Centrifugation at 10000 g was performed on emulsions prepared at 3g/L with 20 mM NaCl and pH 5 and the same monomodal droplet size distribution as before centrifugation was maintained. In addition, samples were stored for one week at 4°C, 20°C, and 40°C, in sealed tubes in order to monitor their stability. The droplet size distribution showed no variation,
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revealing the very high stability of these ChiNC-stabilized emulsions (Figure in SI-2) generally associated to colloids stabilized emulsions.
Effect of concentration of ChiNC on the drop size – When the concentration of ChiNC is increased, as demonstrated in Figure 2 for an aqueous phase at 5 g/L compared to 3 g/L, the emulsion volume increases up to fill the complete volume. The contribution of the droplet size was monitored by measuring the surface mean diameter D(3,2) while varying concentrations of ChiNC in the aqueous phase for a fixed amount of oil. Results are given for a ratio of 20/80 (O/W) (Figure 4). Stable droplets with larger sizes were measured at the lowest concentrations and the size decreased to a diameter of 4.5 µm at 5 g/L of ChiNC in the aqueous phase. This coalescence previously reported for other Pickering emulsions38,39 is based on a limited coalescence process.40,41 This occurs when sonication produces small droplets, i.e. larger areas of oil–water interface than can potentially be covered by the nanocrystals. When sonication is stopped, the partially unprotected droplets coalesce until the interface is sufficiently covered. The droplet diameter can then be modulated with the weight of ChiNC (Figure 4). This is in agreement with the irreversible adsorption of the ChiNC at the oil / water interface. At higher concentrations, this diameter reaches a plateau value at which a denser layer is accompanied by an increased stability of the droplets.
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Figure 4: Average diameter (µm) vs. ChiNC concentration in the aqueous phase (g/L)
As a result, ChiNC proved to bend at the oil/water interface, as illustrated by the SEM images on Figure 5. The coverage of the beads is defined as the ratio of the particles area available to stabilize the interface, and the calculated total area of the oil droplets taking the D(3,2) droplet diameter measured for each emulsion into account.24,
42
A value of approximately 100%
coverage was calculated, revealing the high coverage ability of these solid particles. As can be seen in Figure 5, some crystals overlap, leading to a rigid armored steric protection that inhibits coalescence responsible for their exceptional stability.
1 µm
250 nm
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Figure 5: SEM images of polymerized styrene/water emulsions stabilized by chitin nanocrystals
Pure ChiNC Pickering high internal phase emulsion with modulated cell diameter – High internal phase emulsions (HIPE) are emulsions with an internal phase volume (φ) above 74%.25, 26 The droplets in these highly concentrated emulsions can possibly share interfacial particles within a continuous scaffold,28 provided the particles can be bound or locked together in a continuous structure. Adding hexadecane to the initial emulsion under stirring with a double-cylinder-type homogenizer, resulted in a new gel-like structure with internal phase volume ratios up to 96% (Figure 6). There are generally two ways to reach such a high percentage: (i) increase the polydispersity of the droplet diameters, filling the holes between the droplets; and (ii) deforming the droplets in order to decrease the empty inter-droplet space. In the case of ChiNC, both conditions contribute to the high internal phase ratio. This surprisingly stable nanometric structuration where deformation of the droplets appears without coalescence was already observed with CNC.28 However, using CNC a two-step process was required, including a first emulsion preparation followed by a second step during which oil was added. In contrast, ChiNC may produce similar highly stable gel structure directly from the initial ChiNC suspension. This suggests that less energy was required to stack the nanocrystals at the interface. This could be attributed to the less hydrophilic N-acetyl group in C2 position of the anhydroglucose where cellulose has a hydroxyl function. The same maximum internal phase volume fraction was obtained using the direct or indirect method (Figure 6) but a slight difference was observed between 50% and 75% of internal phase and a stronger deformable visco-elastic gel was formed when using the two-step 12 ACS Paragon Plus Environment
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process. This is attributed to the sonication used in the two-step process. Ultrasonication, providing a high energy, implies a better dispersion of the ChiNC in suspension when the emulsion is prepared. This might lead to a higher surface coverage with more individualized ChiNC and may allow a better structuration of the interfaces.
Figure 6: Percentage of internal phase obtained for ChiNC at 3 g/L in the aqueous phase with 20 mM NaCl and 0.01 mM HCl using a two-step process for which a first emulsion is prepared before introducing the rest of the oil and a direct process for which the oil is directly included to the suspension of ChiNC.
The resulting emulsions prepared with a two-step method are shown Figure 7 according to both the ChiNC concentration and the NaCl concentration, poured directly from the tube onto glass cups. These two parameters revealed the same tendency to increase the structuration of the gel. At low concentrations and low ionic strengths, a soft gel is obtained that can flow on a surface but remain cohesive. Increasing either ChiNC concentration from 1.5 g/L to 3 g/L, or salt concentration from 50 to 100 mM, the gel no longer spreads out on the glass but 13 ACS Paragon Plus Environment
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maintains its initial form. The gel can then be cut into slices that have a solid visco-elastic gel texture.
1.5 g/L
3 g/L
100 mM
50 mM
Figure 7: Emulsion gels formed in various conditions of ChiNC concentration and ionic strength.
The initial droplet sizes were identical as demonstrated figure 3. Also the same percentage of oil was stabilized for each emulsion gel whatever the ionic strength conditions (Fig. SI-3). However, figure 7 shows that the addition of salt strengthens the walls, forming a denser texture. The gel strength of the emulsion gel can then be easily monitored according to ionic strength and ChiNC concentration (Fig. SI-4).
Since the ChiNC are irreversibly adsorbed at the interface, this gel formation is explained by variation of the droplet diameters. When oil is added there is no particle desorption but a swelling of the droplets. Concentrated emulsions can thereby be formed without coalescence leading to a gel. In order to visualize it, emulsions were prepared using cyclohexane, freezedried and metalized with platinum for SEM visualization.
31
The internal structuration
variation with the added oil phase was monitored up to 64% of internal phase, revealing the
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swelling of the droplets. This procedure is similar to those observed. The initial droplets with a diameter of less than 10 µm, as described in the first part of this work, disappeared in favor of a population of larger-size droplets of several tenths of a micron in diameter (Figure 8). The starting concentration in ChiNC in the aqueous phase was 3 g/L, which corresponds to 2.4 g/L considering the whole emulsion (80% of aqueous phase / 20% of internal phase). In the structures presented on the SEM images where additional oil is included, it reaches 36%, 54% and 64% of internal phase, corresponding to amounts of ChiNC of 1.9 g/L, 1.4 g/L and 1.0 g/L of emulsion, respectively. This means that such a structured gel or cellular foam can even be obtained at 0.1% of ChiNC without any additional surface-active material. Furthermore, it demonstrates the strength of such internal structuration since no collapse occurred while drying.
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Figure 8: SEM images of solid emulsions from freeze-dried cyclohexane-in-water emulsions stabilized by ChiNC, to which cyclohexane is added up to the percentage of internal phase indicated. Three different magnifications for each sample are shown (x 100, x 400, and x 1000 or x 2000).
CONCLUSIONS It appears that ChiNC can be used as colloidal particles capable of very efficiently stabilizing interfacial areas and produce highly stable oil-in-water Pickering emulsions. Furthermore, their high stability makes it possible to introduce a large part of internal phase to produce high internal phase emulsion (HIPE) up to a percentage of hydrophobic internal phase as high as 96%. This HIPE produces a gel whose texturing depends on the ionic strength, pH and concentration. It is also easy to handle since, compared to what was observed with cellulose, such emulsions might be produced according to a one-step process where the constituents are mixed directly. As a result, these emulsions that use unmodified ChiNC proved to be in a same time very stable and versatile enough to be modulated from liquid to gel texture. Furthermore, the emulsion is also able to form stable solid foams when both liquid phases are removed using the freeze-drying process without destructuration of the walls of the emulsion. These cellular foams show that the cell dimensions might vary with the oil added to the system.
AKNOWLEDGMENTS The authors would like to thank Joelle Davy for SEM imaging and Nicolas Stephan for SEM assistance (IMN, Nantes, France), Brigitte Bouchet for her assistance in TEM experiments
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(BIBS Platform, INRA Nantes, France) and the Pays de la Loire Regional Council (Matières Network) for financial support.
SUPPORTING INFORMATION Data for surface charge density determination, stability to temperature, effect of ionic strength on the percentage of internal phase and the comparison of the internal phase ratio according to different parameters for HIPE formation is available in SI document. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author * E-mail address: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.
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Table of Contents Graphic
Emulsion gel
Liquid emulsion
Oil > 90%
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