Article pubs.acs.org/Biomac
Modulation of Cellulose Nanocrystals Amphiphilic Properties to Stabilize Oil/Water Interface Irina Kalashnikova, Hervé Bizot, Bernard Cathala, and Isabelle Capron* INRA, UR1268 Biopolymeres Interactions Assemblages, 44316 Nantes, France S Supporting Information *
ABSTRACT: Neutral cellulose nanocrystals dispersed in water were shown in a previous work to stabilize oil/water interfaces and produce Pickering emulsions with outstanding stability, whereas sulfated nanocrystals obtained from cotton did not show interfacial properties. To develop a better understanding of the stabilization mechanism, amphiphilic properties of the nanocrystals were modulated by tuning the surface charge density to investigate emulsifying capability on two sources of cellulose: cotton linters (CCN) and bacterial cellulose (BCN). This charge adjustment made it possible to determine the conditions where a low surface charge density, below 0.03 e/nm2, remains compatible with emulsification, as well as when assisted by charge screening regardless of the source. This study discusses this ability to stabilize oil-in-water emulsions for cellulose nanocrystals varying in crystalline allomorph, morphology, and hydrolysis processes related to the amphiphilic character of nonhydrophobized cellulose nanocrystal.
1. INTRODUCTION Emulsions basically consist of two liquids stabilized by surfaceactive compounds for which most formulations use either surfactant or surface-active polymers. However, it is now well established that solid, colloidal-size particles may be irreversibly anchored at the oil−water interface to form the so-called Pickering emulsions.1,2 They typically require an interfacial solid material that exhibits affinity for the two phases of the emulsion, that is, an amphiphilic character.3−6 In addition to their mechanical properties, Pickering emulsions present the double advantage of being extremely stable and requiring a very small quantity of particles. Research efforts are currently focused on the development of environmentally friendly renewable materials.7−9 The use of solid cellulosic particles with their inherent renewability and sustainability, in addition to their low carbon footprint and low density, is therefore of particular interest for various applications such as cosmetics, pharmaceutics, or medical implants.10 It has long been established that stable nanoparticle suspensions could be prepared by submitting native cellulose to a harsh acid hydrolysis.11 The most widely used process, sulfuric hydrolysis, has been successfully applied to a wide range of native cellulose from a wide variety of plants, microorganisms, and animals and, especially, to cotton linter that leads to nanocrystals (CCN) organized in a Iβ rich crystal lattice. They have been the subject of numerous reports found in the literature.12,13 Concentrated sulfuric hydrolysis favors defibrillation as well as removal of the amorphous or less ordered regions embedded within cellulose microfibrils, leading to highly crystalline particles.14−16 This step generally results in substantially charged surfaces that enhance their hydrophilicity.15,17,18 The study of charge density of cellulose nanocrystals19 has revealed that H2SO4-treated samples result in strong acid surface charges ascribed to sulfate ester groups. © 2011 American Chemical Society
Thereafter, the repulsions due to negatively charged surfaces allow the formation of stable colloidal suspensions. The stability of these suspensions depends on the dimension, size polydispersity and surface charge density. Other sources can be used, including bacterial cellulose that grows as an entangled network of independent nanofibrillar ribbons, which present limited contaminants or other byproducts and are generally obtained in the wet state. Bacterial cellulose nanocrystal (BCN) can therefore be obtained by a milder hydrochloric hydrolysis without preliminary or combined defibrillation because they occur in the form of a rather loose network, contrary to most plant materials. The HCltreated sample results in a weakly charged surface ascribed to carboxyl groups.19 In this case, because the nanocrystals are almost devoid of charge, the apolar nature of the crystals leads to flocculation and less stable dispersion. Moreover, bacterial cellulose is known to be organized in a Iα rich crystal lattice and composed of ribbon-like microfibrils.20,21 It might then expose different surface ratios compared to the cotton nanocrystal, which would make a difference in terms of accessible interfacial area. In a recent study, we showed that it was possible to obtain highly stable oil-in-water Pickering emulsions stabilized by unmodified quasi-neutral bacterial cellulose nanocrystals (BCN).22 However, when sulphated cotton cellulose nanocrystals (CCN) were used, no emulsion was observed. A clear phase separation occurred and all the nanocrystals remained dispersed in suspension in the water phase, whereas the oil was floating on top of the emulsion as a layer of pure oil. Thus, one or several parameters among the source, the allomorph, the morphology, and the surface chemistry of the cellulose nanocrystals appeared to be of prime importance to stabilize an interface. If the charge density Received: November 13, 2011 Published: November 30, 2011 267
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10000 g for the first run up to 75000 g (Beckman Avanti J-30I centrifuge) to obtain a weakly sedimenting colloidal dispersion. Finally, the material collected was dialyzed to neutrality and residual electrolytes were removed with a mixed bed ion-exchange resin. Such a method requires a very limited amount of sulfuric acid per g of dry cellulose and limits degradations. 2.2.5. Desulfation of the Postsulfated BCN. The desulfation of the postsulfated BCN was performed using a procedure similar to CCN desulfation. A 5 N HCl volume was added to an equal volume of 2.2% postsulfated BCN, heated for 3 h at 100 °C, and washed by centrifugation at 6000 rpm for 5 min, and this was repeated six times. Residual electrolytes were removed using mixed bed ion-exchange resin. 2.2.6. Transmission Electron Microscopy (TEM). A total of 20 μL of a cellulose nanoparticles suspension at 1 g/L in water was deposited on freshly glow-discharged carbon-coated electron microscope grids (200 mesh, Delta Microscopies, France) for 2 min, and the excess water was removed by blotting. The sample was then immediately negatively stained with uranyl acetate solution (2% w/v) for 2 min and dried after blotting at 40 °C just before observation. The grids were observed with a Jeol JEM 1230 TEM at 80 kv. 2.2.7. Conductometric Titration. Conductometric titration was performed to determine the surface charge density. A total of 50 mL of a cellulose suspension at 1 g/L in water was stirred under nitrogen for 10 min. Titration was performed with freshly prepared 2 mM NaOH with a TIM900 titration manager and a CDM230 conductimeter equipped with a CDC749 titration cell (Radiometer, Denmark). The apparent degree of substitution was calculated assuming that only one hydroxyl per anhydroglucose could be monosubstituted, leading to the degree of substitution (DS) given by the following equation:
variation has already been studied for the formation of cholesteric liquid crystal organization,12,23 none considered the impact on the wettability of these nanocrystals at an oil/water interface. This work intends to use this original ability of the nanocrystals to stabilize the oil/water interface to study the influences of surface charge on their amphiphilic properties.
2. MATERIALS AND METHODS 2.1. Materials. Traditional nata de coco was bought at a local market in San Jose (Central Luzon, Philippines). All reagents were of analytical grade (Sigma-Aldrich) and water was purified with the MilliQ reagent system (Millipore). Hexadecane was purified by extensive extraction with water to remove most of the contaminating surfactants. For deionization, batch ion exchange was performed using mixed bed resin TMD-8, also provided by Sigma-Aldrich. 2.2. Methods. 2.2.1. Sulfated Cellulose Cotton Nanocrystal (CCN). Whatman filters (25 g, grade 20Chr) were cut into small pieces and mixed with 700 mL of distilled water in a Waring blender until a homogeneous dispersion was obtained. Excess water was removed by filtration. The resulting pulp, kept in an ice bath, was progressively suspended in 65% sulfuric acid so as to reach a final concentration of 61% and subsequently brought to 72 °C under stirring for 30 min. The color of the suspension changed within this period from milky to ivory. After hydrolysis, the suspension was washed by repeated centrifugations at 8000 rpm for 15 min until the supernatant started to become turbid and subsequently diluted by two volumes of water and dialyzed to neutrality for 3 days against water. The residual electrolytes were removed with ion-exchange resin for 4 days at 4 °C. The final dispersion referred as cotton cellulose nanocrystal (CCN) was filtered on a G4 sintered glass, sonicated for 15 min (ultrasonic processor XL 2020, Misonix, NY, U.S.A.) and stored at 4 °C. 2.2.2. Desulfation of the Sulfated CCN. The desulfation of the sulfated CCN was performed by mild acidic treatment. A total of 5 mL of 5 N HCl was added to 5 mL of 13 g/L sulfated CCN dispersion in a sealed vessel and heated for 1, 2, 5, or 10 h at 98−100 °C under stirring. Alternatively, 5 mL of 10 N trifluorohydric acid (TFA) instead of HCl were heated for 10 h at 80 °C under stirring. The resulting suspensions were rinsed and dialyzed as previously described. 2.2.3. Bacterial Cellulose Nanocrystal (BCN). BCNs were prepared according to a previously described protocol.22 In brief, nata de coco cubes were ground in a Waring blender at full speed with ice cubes added to improve impact efficiency. The resulting slurry was filtered and resuspended in 0.5 N NaOH and stirred in a closed flask for 2 h at 70 °C. Alkali were removed by rinsing with distilled water to neutrality. Bleaching was then conducted twice with an 8.5 g/L NaClO2 solution in sodium acetate buffer (pH 4.5) at 70 °C for 2 h. The bleached cellulose was rinsed and hydrolyzed, first in 2.5 M HCl heated under reflux for 1 h, and this treatment was repeated a second time for 30 min like in Gilkes et al.24 After hydrolysis, the suspension was extensively washed to neutrality and further homogenized by a mild sonication for about 1 min (ultrasonic processor XL 2020, Misonix, NY, U.S.A.). After the treatment, the resulting ∼1% suspension was extensively dialyzed against water and deionized with mixed bed ion-exchange resin. For long-term storage, a drop of CHCl3 was added to the suspension. The yield calculated on a dry matter basis was 78%. 2.2.4. Postsulfated BCN. More details can be found in the Supporting Information (SI), but in brief, postsulfation was carried out following a protocol inspired by Chauvelon,25 by reconcentration of initially diluted sulfuric acid. BCN dispersed at 1.34% in water was mixed with 2.2 N H2SO4 (3/2 ratio, v/v) under intensive stirring at room temperature so as to reach 1.5 N, and recovered after centrifugation (6000 g/5 min). The material was mixed with glass beads (diameter 3 mm) so as to spread the nanocrystals over their surface and thereby increase the exposure to gaseous exchange. Glass beads coated with sulfated BCN were dried for 6 h at 40 °C. The beads were subsequently equilibrated over MgCl2 saturated salt solutions in a desiccator and maintained to incipient boiling at 50 °C overnight. Sulfated BCN were recovered by washing the beads with water over a sieve and repeatedly centrifuged for 20 min from
=(Veq × CNaOH × Mw )/m Mw =162/(1 − (D Mw × Veq × CNaOH)/m)
DS
where Veq is the amount of NaOH in mL at the equivalent point, CNaOH is the concentration (mol/L), Mw is the average molecular weight of one sugar unit according to the substitution degree, and m is the weight of titrated cellulose. DMw corresponds to the difference between the molecular weight of a charged and unsubstituted anhydroglucose. For cotton, the difference between sulfated and nonsulfated anhydroglucose units was 80 g/mol, and for bacterial cellulose, the difference between carboxylated and noncarboxylated glucose units was 44 g/mol. The surfacic degree of substitution (DSs) was calculated according to an expression adapted from Goussé et al.,26 taking into account the distance separating two sugar units on the basal area (0.532 nm) and on the flank (0.596 nm),27 assuming a parallelepidic cross-section with average dimensions taken from the TEM and AFM for width and thickness, respectively. Subsequently,
DSs =DS/0.25 for CCN DSs =DS/0.23 for BCN Since the average surface area of one sugar is 0.28 nm2, the results are given as an average amount of elementary charge per nm2 (e/nm2). In the case of samples with a small amount of charge, titration curves showed a very small initial decrease in conductivity when NaOH was added. Several measurements were therefore performed where HCl and NaCl were added before measuring and compared to confirm the results obtained. 2.2.8. Zeta-Potential Measurements. The electrophoretic mobility of aliquots of aqueous nanocellulose suspensions at 0.1 g/L was measured in triplicate with a Malvern 3000 Zetasizer NanoZS, (Malvern Instruments, U.K.). 2.2.9. Emulsion Preparation and Characterization. The oil-inwater (o/w) emulsions were prepared using hexadecane and a nanocrystal aqueous suspension at the required concentrations without further dilution to match an oil/water ratio of 30/70. Practically, 0.3 mL of hexadecane were added to 0.7 mL of aqueous suspension in a plastic vial and sonicated at the 1.5 power level (3 mm 268
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titanium probe, 2 mm below the surface). More precisely, the power level is equivalent to 2 W/mL applied energy (determined by heat balance) and is applied by alternating 3 s sonication with a 3 s standby for 20 s. Droplet diameters were measured by laser light diffraction using a Malvern 2000 granulometer apparatus equipped with a He−Ne laser (Malvern Instruments, U.K.) with Fraunhofer diffraction. The stability of the generated emulsions was checked by centrifugation for 10 min at 4000 g and the thickness of the creaming layer was measured with a digital caliper and converted to volume. Photographs of the vials containing the emulsions were taken with a P1 digital camera (Olympus). Scanning electron microscopy (SEM) images were prepared as previously described22 from styrene/water emulsions performed by sonication before polymerization. Dried beads were metalized with platinum and visualized with a JEOL 7600F instrument. 2.2.10. Wide-Angle X-ray Scattering (WAXS). Cellulose suspensions at 1−3 wt % were solvent-exchanged with acetone and introduced into 1 mm Mark tubes (Hilgenberg GmbH, Malsfeld, Germany) and allowed to dry slowly (50 °C). The dehydrated material was finally compacted in the tube. Transmission WAXS diffractograms were recorded with a Bruker D8 Discover diffractometer (Madison, WI, U.S.A.) using Cu Kα1 radiation (λCuKα1 = 1.5405 Å) produced by a sealed tube at 40 kV and 40 mA, selected using a Göbel mirror parallel optics system, and collimated to produce a 500 μm beam diameter. The samples were exposed with their length parallel to the vertical axis of the detector and orientation was determined by azimuthal integration. The diffracted beams were collected on a two-dimensional GADDS detector (general area detector diffraction system) and the recording time was extended to the maximum available count (107). The diffraction peaks were fitted with pseudo-Voigt peak functions, assuming a linear background. The average dimension of the crystal perpendicular to the diffracting planes with hkl Miller indices, Dhkl, was evaluated using Scherrer’s expression:
prepared. Compared to the values ranging from 0.155 to 0.41 e/nm2 found in the literature for cotton nanocrystals,12,23 this value represents a moderately sulfated sample. However, conversely to neutral bacterial cellulose nanocrystals (BCN), when an emulsion was performed using hexadecane and water, no emulsion could be produced. Therefore, to investigate the electrostatic repulsion contribution, a mild HCl hydrolysis was used to modify the surface chemistry and prepare CCN with a lower sulfate content on the surface.30 After such a treatment, a stable emulsion with CCN was obtained as well as the one already prepared with BCN. Hexadecane and styrene presented similar surface tension (27 and 32 mN/m, respectively) and the same ability to produce stable emulsion. Therefore, solid beads were performed by producing styrene/water emulsions followed by polymerization of the styrene. The same drop size and polydispersity were obtained; we therefore considered this method relevant for visualization. SEM images were then made to visualize surface organization in detail. Both BCN and CCN were identically evenly distributed on the surface and curved along the droplets, building an armored layer (Figure 1). The longest dimension of BCN induced a more developed apparent cross-linking network, but the general aspect showed the same wetting character. To be able to modulate the interfacial properties, the surface charge density was then sequentially reduced by submitting the sulfated sample to 2.5 N HCl acidic treatment for up to 10 h of hydrolysis. Surface charge density was evaluated by conductometric titration and the results are reported in Figure 2 and Table 1. An initial rapid decrease of the surface charge density was observed in the early stages of the hydrolysis that slowed down to reach a plateau value of 0.017 ± 0.002 e/nm2 after 2 h of hydrolysis. This is lower than another reported HCl-catalyzed desulfation procedure using pulp at 3.3% and 4 N HCl at 80 °C, for which only a decrease to 0.062 e/nm2 was reached.30 The present procedure using cotton dispersion at 2.2% and 2.5 N HCl at 100 °C appeared more efficient to remove sulfate charges. To check how the HCl treatment can affect the morphology of the CCN, TEM images were used to control the size variation of the nanocrystals. No drastic variation of the morphology was detected since the acicular shape of the crystals had been maintained and only a slight decrease in size was detected (Figure 3). However, an image analysis of the TEM images revealed that acid hydrolysis, in addition to charge reduction, initially reduced the
Dhkl = 0.94λ /β1/2 cos θ
where θ is the diffraction angle, λ is the X-ray monochromatic wavelength, and β1/2 is the peak width at half-maximum intensity.28
3. RESULTS AND DISCUSSION 3.1. Surface Charge Density Variation on Cotton Cellulose Nanocrystal (CCN). CCN was obtained from cotton linters with a standard procedure using sulfuric acid.18,29 When concentrated sulfuric acid reacts with the hydroxyl groups of cellulose, some negatively charged sulfate groups are introduced by esterification, leading to sulfated nanocrystals. Thus, a batch of CCN bearing 0.123 sulfate charge/nm2 was
Figure 1. SEM images of polymerized styrene/water emulsions stabilized by (a) bacterial cellulose nanocrystals (BCN) and (b) cotton cellulose nanocrystals (CCN). 269
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stability test was performed. It consisted of a centrifugation at 4000 g for 10 min. This test accelerated the natural creaming process due to different densities (hexadecane density is 0.82 g/cm3 at 25 °C) and led to a concentrated emulsion under close packing conditions.22 Thus, water is excluded, forcing the droplets to coalesce when the interface is unstable or when the surface coverage is insufficient. Particles in the aqueous phase were introduced in excess compared to the oil/water interface area, to ensure that unstability of the prepared emulsions result from an inefficient adsorption of the nanocrystals rather than a lack of particles. An emulsion performed with the CCN carrying the highest charge density showed a lack of interfacial properties, leading to unstable emulsion. However, when the same test was performed with CCN recovered after 1 h of hydrolysis, that is, for a charge density below 0.03 e/nm2, a stable emulsion was obtained (Figure 4). This result was also revealed by the rapid decrease of the average diameter of the droplets to about 4 μm with decreasing surface charge density, as well as an increasing emulsion volume (Table 1). In fact, when particles stabilize efficiently the interface, little coalescence is observed and the high oil/water interface leads to the formation of small droplets and, therefore, to a larger emulsion volume. Thus, the surface charge density proved to be critical and the lowering of the charge density by the HCl post-treatment enhanced the interfacial properties of the crystals. However, considering the 10 h HCl treatment, a side effect of hydrolysis was finally observed because the length of the particle decreased and, at the same time, the aqueous phase of the emulsions recovered after centrifugation were opalescent. This suggests that a part of the CCN was unable to stabilize the o/w interface and remained dispersed in the aqueous phase. As an alternative to HCl, similar behavior with the same variation in size of the CCN and charge density was obtained after 10 h of hydrolysis with 2.5 N trifluoroacetic acid (TFA). TFA is an interesting route since it is a weaker acid (pKa = 0.5) than HCl (first ionization, pKa = −8), miscible with most organic solvents.31 As a result, a mild hydrolysis (i.e., less than 10 h of hydrolysis) can be used to peel the surface without penetrating the inner part of the crystal. However, for the longest time of hydrolysis, it can be supposed that the material is finally swollen by the acid treatment and that hydrolysis of the nanocrystals might induce the release of hydrophilic material in the aqueous phase that becomes unable to stabilize the emulsions. 3.2. Sulfation/Desulfation Process on Bacterial Cellulose Nanocrystals (BCN). To further investigate the influence of the source and, thus, the morphology and allomorph of cellulose nanocrystals on interfacial properties, we aimed to modify the surface charge density of uncharged bacterial cellulose. BCN were prepared by hydrochloric acid hydrolysis carried out at reflux temperature and inducing the creation of a few weakly charged groups on the surface, which were ascribed to carboxylic groups.15,19 In good agreement with previous work, the surface charge density of the BCN was found to be 0.012 e/nm2, which corresponds to the level of charge of the lowest sulfated CCN samples. To evaluate the effect of the charge density on the BCN ability to stabilize the oil/water interface, uncharged BCN were allowed to react with sulfuric acid via an original controlled postsulfation treatment to prepare sulfated-BCN (s-BCN). Because BCN are particularly sensitive to H2SO4-catalyzed hydrolysis, BCN were first adsorbed on glass beads before a concentrating process of the acid was carried out by a control of the relative humidity. The acid
Figure 2. Surface charge density vs time of hydrolysis with 2.5 N HCl (solid circles) or 5 N TFA (open circles).
Table 1. Characterization of the Cotton (CCN) and Bacterial (BCN) Nanocrystals Used in Terms of Size and Surface Charge Density of the Nanocrystals and the Resulting Average Diameter of the Droplets and Emulsion Volume after Centrifugation sample CCN T0 CCN 0.25 h HCl CCN 0.5 h HCl CCN 1 h HCl CCN 2 h HCl CCN 5 h HCl CCN 10 h HCl CCN 10 h TFA BCN s-BCN H2SO4 Ds-BCN 3 h HCl
length (nm)
diameter of the emulsion width surface charge emulsion droplets volume 2 (nm) density (e/nm ) (μm) (μL)
189 189
13 13
0.123 0.051
unstable unstable
0 0
189
13
0.033
14.5
61.3
157
13
0.026
7.6
80.2
147
13
0.017
4.2
94.3
141
13
0.018
4.3
84.9
117
13
0.019
3.8
70.8
128
13
0.015
3.4
66.0
855 644
17 16
0.012 0.037
4.8 18.6
108.5 61.3
624
16
0.009
7.2
99.1
length of the particles without changing the width (Table 1). According to the model proposed by Dong et al.15 and ElazzouziHafraoui et al.,15,18 the acid preferentially diffuses into the noncrystalline portions of cellulose and hydrolyzes the accessible glycosidic bonds. Considering the crystalline nanosized particles, most of the amorphous parts were already removed during the first sulfuric hydrolysis. In the early stage of hydrolysis, the sharp decrease in the length of the nanocrystals is therefore attributed to the glycosidic bonds accessible at the extremities. After that, further reaction occurred much more slowly at the extremities and surface of the residual crystalline regions. In that case, the global surface area of the nanocrystal should not considerably change except for the charge density variation. HCl hydrolysis may therefore have a polishing effect, removing the charges present on the surface and free chains at the extremities, without modifying the overall crystalline organization. In order to evaluate the interfacial stabilizing properties of the nanocrystals with the surface charge density, several emulsions were prepared using the modified CCN, and a 270
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Figure 3. Morphology evolution during desulfation by HCl acid hydrolysis by TEM of negatively stained dilute suspensions of CCN. Time of hydrolysis and acid used are indicated in each image.
Figure 4. Stability test (centrifugation at 4000 g/10 min/20 °C) applied to hexadecane in water emulsions stabilized with CCN varying in acid hydrolysis time (hour) and surface charge density (e/nm2).
HCl-desulfation of the s-BCN later decreased the surface charge density to 0.09 e/nm2, indicating that all the sulfated charges were removed. A possible variation of the dimensions of the nanocrystals during the surface modification treatments was monitored by TEM (Figure 5). The morphology of the original BCN was found to be in good agreement with other previous descriptions.13,32 The widths of the nanocrystals were measured by TEM at 17 nm for BCN whereas 13 nm was determined for CCN (Table 1) and it has been reported an identical thickness of 7 nm for both nanocrystals.18,22 BCN is then 4.5 times
concentration was therefore reached without going beyond the critical concentration at which the material changes to a dark color, indicating side reactions and leading to unwanted degraded material. To demonstrate the role of the charge at the surface, we also investigated the reverse reaction. A desulfation reaction of the resulting s-BCN was then carried out with HCl, which yielded to the desulfated BCN sample (ds-BCN). The surface-modified samples were characterized by conductometric titration and the resulting surface charge density calculated (Table 1). As a result, H2SO4-catalyzed sulfation made it possible to increase the surface charge density of BCN from 0.012 e/nm2 to 0.037 e/nm2. The 271
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Figure 5. Morphology evolution of bacterial cellulose nanocrystals (BCN) during post-treatments followed by TEM of negatively stained dilute suspensions of original BCN (a, b); BCN after sulfation (c, d); and BCN after desulfation (e, f) at two magnifications.
longer than CCN. After the sulfation step, the length, as measured by image analysis (Table 1), was decreased from 855 to 644 nm without clear variation of the width. On the contrary, no drastic length variation was observed after the HCl-catalyzed desulfation. This confirmed that sulfuric acid tended to penetrate the crystals, decreasing the length, whereas the hydrochloric acid tended to peel the surface. Consequently, we assume that the morphology was conserved. Nevertheless, the resulting variation in charge densities was enough to induce a variation in the Pickering emulsion formation (Figure 6). Sulfated BCN (s-BCN) presented an increased hydrophilic surface leading to a relatively low interfacial stability because some part of the sample was found dispersed in the aqueous phase instead of being adsorbed at the oil/water interface. After the second post-treatment using HCl-catalyzed hydrolysis to remove the sulfate groups (ds-BCN), it can be seen on Figure 6 that the aqueous phase is transparent, indicating that the initial emulsion conditions without dispersion in the aqueous phase were recovered. This is also confirmed by the increased size of the droplets and the decrease of the emulsion volume (Table 1). The most highly charged nanocrystals formed few larger droplets, whereas
Figure 6. Stability test (centrifugation at 4000 g/10 min/20 °C) applied to hexadecane in water emulsions stabilized with native BCN or after two successive post-treatments with H2SO4 (s-BCN) and HCl (ds-BCN).
uncharged nanocrystals were able to stabilize a large surface area of the interface, revealed by the smaller drop sizes. This confirms the results already shown with CCN that above 0.03 e/nm2, cellulose nanocrystals proved unable to stabilize at the oil/water interface. This process also made it possible to recover the original conditions after simple desulfation with mild hydrochloric hydrolysis that induced a peeling effect, without affecting the inner part of the crystals. A clear chemical modification was observed at the surface without drastic 272
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crystalline planes with sulfate groups and some crystalline planes without charges oriented toward the oil phase. 3.4. Crystalline Orientation at the Oil/Water Interface. Cellulose and cellulose nanocrystals, in particular, are usually considered as hydrophilic matter and are not prone to spontaneously adsorb at the oil/water or air/water interface. Thus, if the macroscopic properties are extrapolated to isotropic nanoparticles, our observations appear to be surprising. The understanding of such behavior makes it necessary to evaluate the extent of edge truncation exposure in crystals, that is, the part of β-linked anhydroglucoses laying flat so as to expose the axial hydrogen, whereas all hydroxyls are equatorial. Structural studies already reported a partial hydrophobic character of cellulose.33,34 As an illustration, one can compare to the amphiphilic character of α-(1−4)-linked glycosidic chains of amylose or cyclodextrins, which allow complexation with apolar small molecules such as iodine, fatty acids, or drugs when adopting appropriate conformations. In fact, several studies have been based on the interactions with hydrophobic sites exposed on cellulosic surfaces, for example, the modeling study of aromatic compounds,35 direct dyes used as molecular probes to characterize cellulose substrates,36 or dyes such as calcofluor that may intercalate within the lattice.37 An even better example is the identification of genetically conserved aromatic amino acids involved in some fungal carbohydrate binding modules that may stack at their flat exposed surface onto “hydrophobic” surfaces.33,38 These experiments tend to demonstrate that some parts of the nanocrystals expose less polar groups at the surface. This character has been attributed to one crystalline plane for which axial CH moieties are directly exposed at an edge truncation at the surface of the nanocrystals. Several WAXS diagrams were recorded from the CCN and BCN samples used before and after surface modification. Two CCN samples were measured: the original sulfated nanocrystal and its desulfated analogue by a 5 h HCl-catalyzed hydrolysis, and three BCN samples: the original neutral BCN, the sample obtained after sulfation (s-BCN), and the one obtained after further desulfation (ds-BCN) by a 3 h HCl-catalyzed hydrolysis. For each sample, four main peaks were visible at diffraction angles of 14.4, 16.7, 20.3, and 22.6°, respectively (see SI), assigned to well-known crystallographic planes indexed according to Sugiyama et al.39 This resulted in eight crystallographic planes indexed according to the surface exposed and the allomorphs (Iα or Iβ). However, some equivalent planes exist between Iα and Iβ allomorphs that are similar in terms of hydrophilic affinity, roughness, and surface energy.34 The various surfaces can therefore be divided into three families (Figure 8). (i) The most exposed family in terms of surface exposure contains
morphological variation. We can therefore deduce that the crystalline lattice as well as the morphology do not have an impact on the ability of the cellulose nanocrystals to stabilize at the oil/water interface. Only the charge density appeared to be a sensitive parameter. 3.3. Counterions and Charge Distribution. When the surface charge density was above 0.03 charge/nm2, regardless of the source (cotton or bacterial cellulose), it was not possible to obtain a stable emulsion in water. This limit in stability was checked for various emulsification processes (rotor stator or sonication) and whatever the energy submitted and the time applied. To understand whether this was due to chemical affinity (hydrophilic/lipophilic balance) or crystal accessibility, the most charged CCN were dispersed in the aqueous phase with ionic strength by the addition of NaCl. In that case, the sulfoester charges are still present, but by screening the electrostatic forces, it suppresses the repulsive interactions. This is in opposition to the desulfated samples for which large parts of bare crystals might be induced by the peeling process. It might discriminate the impact of charge distribution at the surface from a structural point of view. CCN dispersions varying in NaCl concentration were characterized by measurement of both the zeta potential and the emulsion volume that remained after centrifugation 10 min at 4000 g. The zeta potential in the absence of salt was negative with a value of −50 mV (Figure 7a). This was expected since the CCN is negatively charged. The value increased, and above 0.05 M of added NaCl, the zeta potential entered the domain of low-charged surfaces above −20 mV, but no aggregation phenomenon was observed. Moreover, the emulsion volume measured after centrifugation already reached a plateau value when 0.02 M NaCl was added (Figure 7b). The same behavior was observed when KCl was added, showing a nonspecific effect of the salt and evidencing that stable emulsion could be obtained with highly charged nanocrystals. As a result, when unscreened charges were involved, the electrostatic repulsions limit the confinement of particles at the interface, inhibiting, by the way, nanocrystal stabilization at the oil/water interface. However, their presence at the surface of the crystals is not a limiting parameter. We can estimate that sulfate groups induce higher hydrophilic affinity and might decrease the affinity for the oil phase, preventing interfacial adsorption even when these charges are screened. Therefore, this ability to stabilize o/w interfaces, even when charges are present, revealed a nonrandom distribution of the sulfate groups at the surface of the crystals. As a result, an orientation of the crystals might exist at the interface, presenting some
Figure 7. Effect of the ionic strength on the CCN (a) zeta potential of the individual nanocrystals vs NaCl concentration and (b) emulsion volume when prepared with increasing ionic concentration (with NaCl, solid symbols; with KCl, open symbols). The lines serve to guide the eyes. 273
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both BCN and CCN. Globally, few differences are observed, the acidic treatments maintained the crystalline organization, that might indicate similar exposure of the hydrophobic edge planes. Furthermore, the hydrophobic edge planes might not bear charges since they are only composed of CH groups, maintaining the hydrophobic surface available. As a result, we assume the amphiphilic character of cellulose I observed for the Iα and Iβ cellulose lattices might rely on this hydrophobic plane (Figure 8). A particular orientation of the hydrophilic glucosidic residues might therefore present the nanocrystals as an amphiphilic material, illustrating the ability of the nanocrystals to stabilize the oil/water interface as long as only weak electrostatic repulsions are present to maintain such a hydrophobic crystalline plane accessible.
Figure 8. Schematic representation of the stabilization of the Iβ cotton cellulose nanocrystals at the oil/water interface, exposing the hydrophobic edge (200) to the oil phase.
4. CONCLUSION The ability of cellulose nanocrystals to stabilize an oil/water interface was used to investigate their surface amphiphilic character. Therefore, two different origins, namely cotton and bacterial cellulose were used. These two cellulose nanocrystals differ in crystalline organization (Iβ allomorph for CCN and Iα allomorph for BCN), morphology (acicular rod and ribbon-like aspect), nature of the charges present at the surface and surface charge density. Their surface charge density has been modulated by various postsulfation/desulfation treatments. It appeared that regardless of crystalline origin, the electrostatic interaction play the major role in the control of the interface. Cellulose nanocrystals with a surface charge density above 0.03 e/nm2 were not able to efficiently stabilize at the oil/water interface, whereas a decreasing surface charge density led to stable emulsions. Furthurmore, this wetting change can be managed by a desulfation process or by screening charge repulsions. It involves clearly an amphiphilic character of cellulose. We assume that the amphiphilic character reside in the Crystallin organization at the elementary brick level. Irrespective to all the parameters investigated, the (200)β/(220)α hydrophobic edge plane appears responsible for the wettability of the cellulose nanocrystals at the oil/water interface, and therefore, its accessibility will deduce the ability to produce stable emulsions.
four hydrophilic and moderately rough surfaces responsible for the major properties, (1−10)β/(100)α and (110)β/(010)α. Their dhkl values are assigned to the means thickness and width of the nanocrystals, respectively.40 The two later families are of minor importance in terms of surface area as they are located at the corners of the cellulosic crystals: (ii) one is flat and hydrophobic (200)β/(220)α, and the last one (iii) (010)β/(110)α that presents rough and hydrophilic surfaces, was not detectable on the spectra. The average crystallite dimensions of each plane were calculated using Scherrer’s equation as applied to individual Voigt peaks that were determined by fitting experimental transmission WAXS powder diagrams (Table 2). The sizes determined are in good Table 2. Crystal Sizes According to the Miller Indices for the Native and Modified CCN and BCN Samples Determined by X-ray Diffractograms Miller indices (1−10)β/ (100)α (110)β/ (010)α (200)β/ (220)α
CCN
CCN 5 h HCl BCN
sBCN
dsBCN
plane characteristics
4.2
4.2
5.7
5.0
5.2
thickness
4.1
3.8
7.6
6.35
6.0
width
6.2
5.9
6.3
5.8
5.9
hydrophobic smooth edge
5. ACKNOWLEDGMENTS The authors thank Mrs. Joann Dar for the purchase of nata de coco, Brigitte Bouchet for her assistance in TEM experiments (BIBS platform, INRA Nantes, France), Nicolas Stephan for his assistance in SEM experiments (IMN, Nantes, France), Joelle Davy for conductometric titration, Bruno Pontoire for the WAXS experiments, Solene Grosbois for her excellent technical assistance, and the Pays de la Loire Regional Council (RMB network) for financial support.
agreement with earlier investigations by other authors.18,40,41 However, these values are lower than the dimensions measured by TEM. For example, for the unmodified BCN, the width was 17 nm by TEM and only 7.6 nm by the X-ray diffraction method. This can be attributed to an overestimation of the TEM values due to both the aggregating drying process and the shadowing effect of the negative staining with uranyl acetate.18 Furthermore, X-ray diffraction can only detect continuous crystalline units. The difference observed between TEM and WAXS techniques strongly suggested that the crystals measured were not monocrystals but an assembly of several individual bricks. Thereby, cotton appeared constituted by squared elementary brick of 4.1 × 4.2 nm, whereas BCN had a more ribbon-like cross section of 5.7 × 7.6 nm. The impact of sulfation/desulfation processes can then be monitored at a crystalline elementary brick level (Figure 8). For both CCN and BCN, the acid post-treatments showed few variation of the crystalline planes. The (1−10)β/(100)α planes giving the thickness remained unchanged. The (110)β/(010)α planes giving the width showed variation only for BCN. Finally, the (200)β/(220)α, giving the diagonal between the more hydrophobic edges, presented about the same mean value for
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ASSOCIATED CONTENT * Supporting Information Additional details about the BCN post-treatment using H2SO4 and the WAXS diagrams. This material is available free of charge via the Internet at http://pubs.acs.org. S
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AUTHOR INFORMATION Corresponding Author *Tel.: +33 (0)2 40 67 50 95. Fax: +33 (0)2 40 67 51 67. E-mail:
[email protected]. 274
dx.doi.org/10.1021/bm201599j | Biomacromolecules 2012, 13, 267−275
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