Structural Description of the Interface of Pickering Emulsions

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Structural description of the interface of Pickering emulsions stabilized by cellulose nanocrystals Fanch Cherhal, Fabrice Cousin, and Isabelle Capron Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01413 • Publication Date (Web): 14 Dec 2015 Downloaded from http://pubs.acs.org on December 28, 2015

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Structural description of the interface of Pickering emulsions stabilized by cellulose nanocrystals Fanch Cherhal1, Fabrice Cousin2, Isabelle Capron*1

1 UR1268 Biopolymères Interactions Assemblages, INRA, F-44316 Nantes, France 2 Laboratoire Léon Brillouin, CEA-Saclay, Gif-sur-Yvette, France

Abstract The cotton cellulose nanocrystals (CNCs) used in this study are rod-like particles with dimensions in the nanoscale (195 nm long, 23 nm width and 6 nm thick) able to stabilize Pickering emulsions. The adsorption of CNCs at an oil-water interface has been investigated by small angle neutron scattering (SANS) with and without surface charge, and varying CNC concentration from 2 to 5 g/L. Average thicknesses of the interfacial CNC layer around the emulsion droplets of 7 nm and 18 nm were determined for charged and uncharged CNC respectively regardless of their concentration in suspension. This suggests that CNC particles lie as a monolayer varying in surface density. Using several phase contrast variations, the neutron wave vector (Q) dependence with the intensity showed that CNCs are in contact with the oil phase only via the surface of the CNC and not immersed in oil since the Porod behavior is observed over the whole Q-range revealing no deformation of the oil surface at a nanometer scale. This result promotes the postulate that the (2 0 0) crystalline plane of the CNC directly interacts with the interface.

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Keywords: Cellulose _ whiskers _ Nanoparticle _ Pickering emulsion_ SANS_ interface.

Introduction

Increasing environmental awareness is prompting scientists and manufacturers to develop strategies for environmental sustainability by using processes and materials with low cost, low energy consumption and low toxicity, together with high biodegradability. In this framework, bio-based nanoparticles are good candidates to stabilize emulsions, replacing commonly used surfactants that are generally derived from the petro-chemical industry and of which only a small fraction is of plant origin.1 This would be of great interest since emulsions are widely used in various application domains such as food, cosmetics, pharmaceuticals and coatings.2, 3 It is known that solid particles of colloidal size can be strongly adsorbed at oil/water interfaces, forming the so-called Pickering emulsions4, 5 or, more generally, solid-stabilized emulsions.6, 7 To summarize the particularity of such emulsions: (i) the adsorption energy at the interface is thousands of times greater than for surfactants; (ii) to adsorb at the interface, the particles have to be partly wetted by both phases; and (iii) surface chemistry is of prime importance since the continuous phase is the one in which the particles are dispersed. As a consequence, they are irreversibly anchored, unlike surfactant molecules that adsorb and desorb within a very short time, providing a solid protection for the droplets. Because of these particular properties, colloidal particles may be efficient species for the long-term stabilization of metastable materials.6, 8, 9 Among the particles generally used, non-spherical nanoparticles (e.g., rods, sheets, wedges, disk-like and needle-like particles, etc.) are rarely mentioned,10-13 whereas they can be more efficient in stabilizing emulsions than spherical ones. For example, the mechanical properties of monolayers as measured by surface shear 2 ACS Paragon Plus Environment

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rheology showed that the monolayer of ellipsoids exhibits a substantial surface modulus, even at low surface coverage, and can be used to create more elastic monolayers compared to aggregate networks of spheres of the same size.14 It forms monolayers with buckling capacity under compression. Furthermore, anisotropic particles can also yield oriented structures.15

Cellulose fibers count among the main available biodegradable and sustainable molecules.1, 16 After hydrolysis, crystalline solid nanometer-sized rod-like particles called cellulose nanocrystals (CNCs) can be obtained from these fibers.2, 17-19 CNCs are composed of a small number of laterally associated elementary crystallites (3–4 for cotton, 2–3 for avicel and tunicin) and their thickness is that of one crystallite.18, 19 Their lateral dimensions range from 4 to 20 nm and their length from 100 nm to several micrometers, depending on the source of the cellulose.10,

18

In the case of cotton, average dimensions were recently measured with

precision in aqueous suspension by Small Angle Neutron Scattering (SANS)20 with values of 195 ± 35 nm in length, 22 ± 3 nm in width and 6 ± 0.2 nm in thickness. Acid hydrolysis with sulfuric or phosphoric acid induces the formation of charged groups at the surface in water, promoting electrostatic interactions and, in turn, providing good colloidal stability21 whereas HCl yields uncharged CNCs. Previous SANS studies carried out on suspensions at 5 to 8 g/L revealed that charged CNCs in suspension are stable at low salinity, but self-assemble forming aggregate with an fractal structure when electrostatic repulsions are screened by salt addition, typically above 10 to 50 mM NaCl. If the surface charges are removed by desulfation, uncharged CNCs aggregate in solution, regardless of the salinity, to form also fractal aggregates. In this latter case, the density of such structures is higher than for charged CNCs.20


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Recent work showed that highly stable oil-in-water Pickering emulsions may be stabilized using unmodified cellulose CNCs that form a dense 2D interfacial network.22,

23

The

adsorption process of the CNCs at the oil-water interface was attributed to the (200) β/(220) α crystalline edge that is more hydrophobic than the faces of the CNC and orients towards the oil phase.23 The properties of these systems in terms of stability and mechanical behavior are due to the irreversible particle adsorption and steric barrier to coalescence.6,

9

It gives

specificity for the drop size control at low particles concentration, where the drop diameter evolution is driven by limited coalescence. 24 At higher concentrations monodisperse droplets of approximately 4-10 µm in diameter are obtained.9,

24

It therefore seems possible to

modulate the porosity of the interface and visco-elasticity of CNC-based emulsions.10 However, it is still not clear the way CNCs adsorb at the interface and the way they assemble to provide a steric barrier that avoids coalescence. The aim of this paper is to probe the organization of the CNCs at the oil-water interface and evaluate the influence of surface charge. Emulsions were prepared from two aqueous CNC suspensions - one with charged CNCs and the other one with neutral CNCs after removing sulfate groups from the surface and then compared at various CNC concentrations. The emulsions are characterized by combining laser light diffraction to obtain droplet diameters and SANS in order to measure the thickness and roughness of the solid shell formed by the CNC at the oil/water interface.

Materials and Methods Materials: All of the reagents used were of analytical grade (Sigma-Aldrich), and water was purified with the Milli-Q reagent system (18.2 MΩ cm Millipore Milli-Q purification system). CNCs were obtained from Whatman filters (grade 20 Chr). A caliper was used to measure the volume fractions of emulsions in tubes.


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CNC preparation and characterization: The two types of cellulose nanocrystals (CNCs) were prepared from cotton linters according to the method of Revol et al.17, with minor modifications as described earlier. Briefly, sulfated CNCs were prepared using sulfuric acid hydrolysis at 58% at

70 °C under stirring for 20 min. A part of the same sulfated CNCs was further desulfated by mild acidic treatment in 2.5 N HCl as previously described.19, 23 After hydrolysis, the suspensions were washed by centrifugation, dialyzed to neutrality against Milli-Q water for one week, and deionized using mixed bed resin (TMD-8). The final dispersion was sonicated for 10 min, filtered, and stored at 4 °C. For conductometric titrations, 10 mL of a CNC suspension in water (0.1% w/v) were titrated with 10-3 mM NaOH using a TIM900 titration manager and a CDM230 conductimeter equipped with a CDC749 titration cell (Radiometer, Denmark). A surface charge density of 0.16 e/nm² was measured for the initial sulfated CNCs. No sulfate was detected for the desulfated CNCs based on the absence of a negative slope, indicating no detectable sulfates, although traces of weak acids may be detected by a short delay of the positive slope.19

Emulsion preparation: The oil-in-water (o/w) emulsions were prepared using hexadecane and a CNC aqueous suspension at the required concentration without further dilution. 50 mM NaCl was added to the sulfated suspension and 5 mM to the desulfated sample to limit repulsions due to charged groups potentially present at the surface, without forcing saltinduced aggregation. For all experiments 3 mL of emulsions were prepared using oil/aqueous phase ratios of 10/90, 20/80 and 30/70, respectively, and sonicated for 5 s with an ultrasonic device with a dipping titanium probe close to the surface (Amplitude 2 corresponds to 4 W/mL applied power), using intermittent pulses. All preparations were oil-in-water emulsions with a milky white color. Hexadecane was chosen because its low solubility in water renders the ripening mechanism by molecular exchange negligible.25 Average droplet diameter was measured by laser light diffraction using a Malvern 2000 granulometer apparatus equipped 5 ACS Paragon Plus Environment

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with a He-Ne laser (Malvern Instruments, U.K). Scanning Electron Microscopy (SEM) images were prepared as previously described (Kalashnikova, Bizot et al. 2013) from styrene/water emulsions performed by sonication and degassed with nitrogen before polymerization at 50°C for 24h. The resulting beads were washed by repeated centrifugation. Dried beads were metalized with platinum and visualized with a JEOL 6400F instrument (IMN-Nantes).

Evaluation of coverage: The emulsions were characterized for their D(3,2) mean Sauter diameter or surface-volume diameter distribution. A relationship between the mass of solid particles (m) and the D(3,2) average radius of the droplets (D) has already been proposed10, 26 where the surface coverage C is given by the ratio of the theoretical maximum surface susceptible to be covered by the particles Sp and the total surface displayed by the oil droplets Sd:

C = Sp

(1)

Sd

where S p

=

=

N p Ll

Sd = 4πR²× 3Voil = 3Voil R 4πR3

m hρ p

(2)

and

(3)

where Np is the number of CNCs, L, l and h are the length, width and thickness of the CNCs, respectively, m is the mass of the CNCs, ρp is the CNC density, R is the average drop radius and Voil is the volume of oil included in the emulsion after centrifugation. Assuming that all the particles are absorbed at the interface, the total coverage (C) i.e., the percentage of droplet surface area covered by cellulosic particles, can be expressed as:

C

=

mD 6hρVoil

(4)

The inverse average droplet diameter (D) can then be written as follows: 6 ACS Paragon Plus Environment

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1

D

=

m

6hρ PVoil C

(5)

Small Angle Neutron Scattering (SANS) experiments were carried out at room temperature on the small-angle PACE spectrometer at the Laboratoire Léon Brillouin (CEA/CNRS) in Saclay (France). Three configurations were used, covering a Q-range from 0.0024 to 0.44 Å-1 where Q is the wave vector (Q= 4π sin θ/2, where θ is the scattering angle and λ is the neutron wavelength). All the emulsions were loaded into quartz cells (Hellma) with small path lengths of 1 or 2 mm. The azimuthally averaged spectra were corrected for solvent, cell and incoherent scattering, as well as for background noise. A systematic fast creaming was observed for all experiments with clear water at the bottom and cream on which beam was focused. Several measurements were duplicated showing that that drop concentration variation might slightly modify the volume fraction of emulsion and thereby the intensity but had no effect on the conclusions. In this three-component system (CNCs, water and oil), three different conditions of neutron contrasts were used where the scattering length densities (SLD) of two components were matched using hydrogenated and deuterated solvents (Fig. 1): (i) 100%D20 and 6.4%C16H34/93.6%C16D34 (SLDwater = SLDoil). In this condition, only the shell of the droplet at the water/oil interface is probed; (ii) 100%D20 and 68%C16H34/32%C16D34 (SLDCNCs = SLDoil); and (iii) 64.5%H20/35.5%D20 and 100%C16D34 (SLDCNCs = SLDCwater). The latter two conditions provide an insight into the roughness of the droplets (from the Porod law) in order to determine if the CNCs are preferentially located in the water or in the oil. The SLD of cellulose was estimated from Jean et al.27 Deuterated solvents were used to reduce incoherent scattering as much as possible. Table 1 summarizes the SLD of the different constituents.

Table 1: Scattering length density (SLD) of the different constituents of the emulsion system 7 ACS Paragon Plus Environment

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Scattering length density Component (Å-2) hexadecane (C16D34)

6.85 10-6

hexadecane (C16H34)

-0.43 10-6 1.9 10-6

cellulose hydrogenated water (H2O)

-0.56 10-6

deuterated water (D2O)

6.38 10-6

a

b

Contrast Shell SLDoil = SLBwater

Contrast Core-shell SLDNC = SLDoil

c

Contrast

Core SLDNC = SLDwater

Figure 1: Concept of contrast variation and contrast-matching, applied to emulsion geometry. Three cases are presented: (a) the continuous phase and droplet phases have similar SLD values; (b) the droplets and the shell have similar SLD values; and (c) the shell and the continuous phase have similar SLD values.

Results and discussion The cotton CNCs usually bear negative charges that originate from the sulfuric acid hydrolysis. These sulfate half-ester groups promote repulsions at the interface, limiting the surface coverage and, consequently, leading to unstable emulsions. These repulsions might be lowered in order to efficiently stabilize drops. Two methods were used to decrease electrostatic repulsions: (i) adding salt to water, with a salinity of 50 mM; or (ii) removing the


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charges at the surface of the CNCs by using an HCl hydrolysis post-treatment leading to desulfated quasi-neutral particles.19, 23

Emulsion characterization with sulfated and desulfated CNCs. Emulsions using dispersions of CNCs at various concentrations were prepared with three different oil/water ratios: 10/90, 20/80 and 30/70. Sulfated and desulfated CNCs were used to stabilize oil/water interfaces, and the resulting emulsions prepared using identical conditions were compared. The diameters resulting from the different ratios could not be superimposed when studied vs. CNC concentration. However, in both cases, the drop diameter vs. the CNC concentration expressed in weight of CNC particles per mL of oil introduced (mp) superimposed perfectly. This unique curve points out that the parameters that tune the droplet diameter are the amount of CNCs and the volume of oil to be dispersed. This is due to the irreversible nature of the adsorption. Indeed, as long as all the CNCs are adsorbed at the oil/ water interface, the volume of water is not involved in the control of droplet size but present as the continuous phase in excess. Figure 2a shows the average drop diameter evolution as a function of the variation of the amount of CNCs for 1 mL of oil phase. The curves for sulfated and desulfated samples follow the same trends: in the low concentration domain, a sharp decrease in the drop diameter is representative of a limited coalescence process9, 24 where the amount of CNCs controls the drop size. In the higher concentration domain, the diameter stops decreasing sharply to stabilize with very similar values for both samples at a value of ~4 µm. This constant value raises the question of the possible presence of free CNCs in the aqueous continuous phase in this regime. This issue was addressed in a previous study10 where we measured the amount of CNCs present in the residual aqueous phase after emulsification and centrifugation by sugar analysis after strong acid hydrolysis. It was null or negligible. We thus infer that the total


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amount of CNC was adsorbed at the interface in all the conditions used in the present study. Consequently, this plateau might be attributed to an inability to decrease the drop size in those conditions. Desulfated CNCs led to slightly bigger diameters than sulfated ones for the same mp. This is better visualized when the same values are plotted in 1/D vs. mp (Fig. 2b). Given that the volume of oil is fixed (Voil = 1 mL) and that there are no free CNCs in the aqueous phase, the slope changes might reveal either a different thickness of the layer at the water/oil interface and/or a different compactness of this layer. The different slopes for both samples reveal a different structure of the interface. In a previous study,20 it was shown by SANS that uncharged CNCs form more aggregated and compact structures than charged CNCs at 50 mM NaCl in aqueous suspension. More desulfated CNCs might then be necessary to stabilize drops of identical diameter. A deviation from the first linear dependence of 1/D vs. mp occurred at higher concentrations. This deviation arises at a critical concentration that is shifted towards higher concentration values (from 7 to 11 mg/mL of hexadecane) when desulfated CNCs are used. Consequently, the presence of charge on the surface might modify the coverage and, as a result, the organization of the interface. 2,500

a

b

2,000

D 10% S 10%

-1

D 30% S 30%

80

1/D (cm-1)) 1/D (µm

100

D[3,2] (µm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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60 40 20

1,500 1,000 500

0 0

5

10

15

20

25

30

35

40

45

mp (mg per mL of hexadecane)

Desulfated CNC Sulfated CNC

0 0

10

20

mp (mg CNC per mL

30

40

of hexadecane)

Figure 2: Two representations of the evolution of the Sauter diameter vs. the amount for sulfated CNCs (S) and desulfated CNCs (D) per mL of hexadecane: (a) D[3,2] vs. mp with squares for 10/90 and triangles for 30/70 oil/water ratio.; and (b) 1/D[3,2] vs. mp.


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b

a

1 µm

1 µm

c

500 nm

e

250 nm

d

500

f

250 nm

Figure 3: SEM images of polymerized styrene-in-water emulsions prepared at 27 mg/mL of hexadecane at three different magnifications for sulfated CNCs (a, c, e) and desulfated CNCs (b, d, f).

As a comparison, SEM images were carried out for both CNC samples in the same conditions (27 mg/mL of hexadecane) (Fig. 3). Both showed drops with similar dimensions and a partial local alignment of CNCs. It appears on these images that desulfated samples form less densely covered surfaces.

Characterization of the interface. SANS experiments were carried out on the emulsions to provide local scale information on the structuration of CNCs directly in situ, at the interface.


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By matching the scattering length density (SLD) of the two liquid phases, only the residual scattering contribution of the interface was observed (see Materials and Methods). Three CNC concentrations were probed. They were chosen above the critical concentration, above the inflection of the D[3,2] vs. the mp curve (Fig. 2) where stable droplets of about the same dimensions were obtained (4.5 µm and 5 µm for sulfated and desulfated samples, respectively). The drop diameters were large compared to the typical spatial scale probed by the SANS (D[3,2] >> 1/qmin). As a result, only a small surface area of the droplets was probed, and the interface appears as a thin 2D flat film.

Figure 4: I=f(Q) SANS curves of CNC suspensions at 7, 18 and 45 mg/mL hexadecane (a) for sulfated CNCs in 50 mM NaCl and (b) for desulfated CNCs in 5 mM NaCl.

Figure 4 shows the curves obtained while increasing the concentration of CNCs from 7 to 45 mg/mL hexadecane (which corresponds to concentrations of 0.8 to 5 g/L in the aqueous phase for a 10/90 oil/water ratio). The resulting interface signal shows a power law decrease of the intensity with the wave vector (Q) as Q-2 in the low Q range, which is characteristic of the scattering of two-dimensional objects. The form factor of this flat film may be modeled by a


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simple geometric model of a thin disc of thickness 2h and a very large radius according to the following equation:28, 29

PሺQሻ ≈

ሺQhሻ2 2 exp ቆ− ቇ ሺQRሻ2 3

Qh ≤ 1 et QR ≫ 1

This is valid when R is very large compared to h.

The thickness value (2h) was directly extracted from the slope of the curve ln(Q2I) as a function of Q2. Charged CNCs in 50 mM NaCl gave an average thickness of the interfacial layer of 7 nm, measured for a 10/90 oil/water ratio emulsion (Table 2). This thickness value is almost the lateral cross section dimension already reported for individual elementary crystals,18,

23

suggesting that individual CNC particles lie as a monolayer that is oriented

according to the (2 0 0) lateral dimension of the crystal and not according to the width generally reported with lower values around 4 to 6 nm.

The slight intensity variation observed from one curve to another on Fig. 4.a is due to a slightly modified emulsion volume for sulfated CNCs. It is indeed difficult to properly estimate the exact volume of emulsion probed by the neutron beam due to sampling and creaming process occurring during preparation of the vials so before neutron measurement. However, the same thickness value of 7 nm was measured, irrespective of the amount of CNC introduced for the sulfated CNCs. As a result, a monolayer of CNC was always measured at the interface, regardless of the CNC concentration.

In order to check if a part of the CNC was desorbed from the interface and released in the continuous phase, a sample was prepared with a mp of 130 mg/mL hexadecane (concentration of 15 g/L in the aqueous phase). This value is considerably higher than the one required for


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interface stabilization since the drop diameter did not decrease proportionally and stabilized at 3 µm, which implies that CNCs are probably still present in the aqueous continuous phase. In fact, the variation of the scattered intensity with Q no longer showed the Q-2 decay observed at lower mp. The scattered intensity is thus the sum of the scattering from the free rod-like CNCs that are present in the continuous phase and of the scattering from the interface forming the 2D films. The thickness could no longer be determined. The same sample was then rinsed by repeated centrifugations and redispersed in heavy water. The decay Q-2 was then recovered, showing that free CNCs were washed away. This washing step resulted in a saturated surface whose scattering curve was precisely similar to the previous ones. The obtained thickness was 8 nm, suggesting the presence of a single monolayer once again. This is additional evidence that, in the conditions considered up to 5 g/L, the CNCs were all adsorbed at the interface because all of the curves revealed a perfect Q-2 decay behavior over the total Q range investigated, without requiring any rinsing step.

Table 2: Characteristics of the samples probed by SANS. The thickness of the cellulosic interface is determined by analysis of the SANS curves and the radius of droplets by laser light diffraction (measurements in triplicate). CNC concentration Mass of CNC

Thickness

Radius

aqueous mp

(nm)

(µm)

in

the

phase. (g/L)

(mg/mL

hexadecane)

Sulfated CNCs 0.8

7

7±2

4.7 ± 0.3

in 50 mM NaCl

2

18

7 ± 0.5

3.4 ± 0.1

5

45

7 ± 0.5

2.1 ± 0.1

15 (before rinsing)

130

Not determined

Not determined

15 (after rinsing)

Not determined

8 ± 1.5

1.5 ± 0.1


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Desulfated

0.8

7

18 ± 0.5

5.0 ± 0.6

CNCs

2

18

19 ± 0.5

3.3 ±0.3

in 5 mM NaCl

5

45

18 ± 0.5

2.8 ± 0.2

Table 2 compares thicknesses of the cellulosic interface obtained with the sulfated CNCs in 50 mM and the desulfated CNCs dispersed in 5 mM NaCl in order to suppress repulsive charges due to sulfate and potentially other than sulfate groups present at the surface, without forcing salt-induced aggregation. For desulfated CNCs, the CNCs arrange differently than sulfated CNCs. A thicker layer of 18 nm is obtained. Since this layer is two to three times the thickness of the CNCs, this indicates that aggregates are deposited on the surface. However, the thickness obtained with desulfated CNCs was constant and independent of the initial CNC concentration in the aqueous phase.

For both samples, at low concentrations, the limited coalescence stabilizes the drop at the minimum coverage required to obtain stable emulsions. Using rods, this minimum coverage might be reached when a stable percolated network forms on the drop surface. Consequently, a full coverage of the surface by CNCs is not necessary to stabilize the droplet. When increasing the concentration, the CNCs present in the monolayer may reorganize, allowing the adsorption of more nanoparticles, thus densifying the interface. In contrast to spheres, the reorganization can be easily explained by a progressive orientation of the rods that fill a large part of the available space at the surface, leading to the local alignments visible on the SEM images.


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The coverage of the surface of the droplets can then be calculated from Equation 4 where the three parameters of interest are the drop diameter that varies with the amount of CNC, the CNC density, and the thickness of the CNC interfacial layer. This equation postulates that the

CNCs are lying flat on the larger surface. It however allows a relative comparison of the samples. As a result, figure 5 shows the drop coverage calculated for both charged and uncharged samples using the thickness values determined by the SANS experiments, i.e., 7 nm for sulfated CNCs and 18 nm for desulfated CNCs. In the low concentration domain, the system is directed by the limited coalescence process. After emulsification, droplets coalesce in order to decrease the surface of interface until a minimum coverage for stability is reached. The drop size stabilizes at the lowest drop coverage required for stabilization. It occurs at a lower drop diameter while increasing the CNC concentration. As a result, fixed values of the coverage are calculated in that concentration domain. This coverage value corresponds to the minimum fraction required to obtain a stable emulsion. For charged CNCs, a value of 85% of the surface coverage with a fixed interface thickness of 7 nm was obtained. This is in perfect accordance with previously published results.10 The neutral CNC with a thickness of 18 nm gave a covered surface percentage of only 45%, revealing a more porous interface. Thicker assemblies are deposited on the interface, leading to less dense layers.


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Desulfated CNC Sulfated CNC

140% 120%

Coverage

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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100% 80% 60% 40% 20% 0% 0

2

4

6

8

10

12

14

mp ( mg CNC per ml of hexadecane) Figure 5: Evolution of the calculated percentage of coverage of the droplet surface with the mass of CNC introduced in the aqueous phase to stabilize 1 mL of hexadecane for sulfated CNCs in 50 mM NaCl and desulfated CNCs in 5 mM NaCl.

Moreover, to adsorb at the interface, particles might be wetted by both liquids. CNCs are hydrophilic particles that easily disperse in water but not in hydrophobic medium.

Insight into the wettability at the interface. Taking advantage of the contrast variation, several configurations were prepared. On one hand, the CNC shell was matched with the aqueous continuous phase using appropriate fractions of hydrogenated and deuterated water On the other hand, the CNC shell was matched with the oil dispersed phase, combining appropriate fractions of hydrogenated and deuterated hexadecane (see Materials and Methods). Figure 6 shows the scattering curves of the emulsions for the two contrasts. For the purpose of comparison, they are compared with the curve of an aqueous suspension of sulfated CNCs in 50 mM NaCl, as measured in a previous study.20 When the aqueous


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continuous phase was matched with CNCs (green triangles), a Q-4 power law decay of the scattered intensity was observed over the complete Q range, revealing a typical Porod behavior of a net interface without roughness. This shows that CNCs are not present in the oily phase; otherwise, deviations from the Porod law would have been observed. Conversely, for the other contrast where the oily phase was matched with CNCs (blue squares), the characteristic Q-4 decay of the Porod behavior is only observed at low Q down to 0.013Å-1, where larger dimensions were probed. At this low Q range, only a general view of the drop interface is probed without details. Going to larger Q corresponds to a change in scale observation. In the intermediate Q range, a clear deviation from the Porod is observed, and the scattered intensity superimposed on the scattered intensity of CNCs when dispersed in aqueous suspension (without any oil phase) at the same ionic strength (red dots). The CNCs are then fully visible in this Q-range. This signature of the entire CNCs was only observed when probing the shell surface whereas no surface deviation of the oil surface was noticed. They appear thus entirely present in the water phase of the emulsion. In summary, there is evidence that the CNCs are in contact with the oil phase only via the surface of the CNC; they are not immersed in oil at the nanometer scale since the Porod behavior is observed over the whole Q-range revealing no deformation of the oil surface.

Figure 6: SANS results of emulsions stabilized by sulfated (S-CNC) in 50 mM NaCl and


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desulfated (D-CNC) in 5 mM NaCl when CNCs are contrast-matched to oil (blue curves) and when CNCs are contrast-matched to water (green curves), compared to the scattering of CNCs in aqueous suspension (S-CNC at 7.8 g/L in 50 mM NaCl and D-CNC at 5.4 g/L in 5 mM NaCl) from the reference 20 (red curves, multiplied by a constant factor).

The Q-4 decay was observed for both sulfated and desulfated samples, regardless of the conditions tested when CNCs are contrast-matched to water. In the same way, when the desulfated CNCs are contrast-matched to oil, the scattered intensity superimposed on the scattering of CNCs in suspension at intermediate Q. This shows that the aggregation process revealed by the increased interface thicknesses from 7 nm to 18 nm (Table 2) did not affect the adsorption mode at the interface, leading to the same smooth surface in all of the cases, irrespective of the particle size. The desulfated CNCs showed larger width by microscopy images19 and a large tendency to aggregation.20 Here a thickness nearly 3 times larger is obtained that can be understood by the compression of wider particles fully wetted by water as schematically illustrated in figure 7.

h = 18 nm

h = 7 nm

Sulfated CNC

Desulfated CNC


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Figure 7: Schematic representation illustrating the flat interface at the oil surface with totally wetted CNCs in the aqueous phase leading to a rough external interface and a thicker interface with desulfated CNCs.

Compared to previous SANS results where an aggregation of sulfated CNCs was measured for suspensions in the presence of salt (from 5 to 200 mM NaCl), the sonication step during the emulsification process allows complete disruption of these aggregates and the formation of a 7-nm-thick shell. After that, the shell formation at the interface preserves the CNCs from further aggregation. On the other hand, the same sonication step led to aggregated structure for the desulfated CNCs, revealing stronger and/or faster CNC-CNC interactions.

CNCs at the oil-water interface. According to wide angle X-ray scattering experiments, it was shown that the elementary unit of cotton cellulose was a square section of approximately 4 nm23 to 6 nm.18 The precise crystal and molecular structure, together with the hydrogenbonding system in cellulose Iβ, have been determined by Nishiyama et al.30 The surface of cotton CNCs can be defined according to various crystalline planes that can be divided into three families. The most exposed family in terms of surface exposure contains four hydrophilic and moderately rough surfaces responsible for the major properties, (1 -1 0) and (1 1 0). Their dhkl values are assigned to the mean thickness and width of the nanocrystals, respectively.31 The other two families are of minor importance in terms of surface area since they are located at the corners of the cellulosic crystals: the (0 1 0) plane presents a relatively rough and more hydrophilic surface; and the last one, the (2 0 0) plane, is flat with CH groups and is more hydrophobic.32 This last one was already involved in several hydrophobic interactions with molecules.23,

33, 34

The present result strongly suggests that the (2 0 0)

crystalline plane of the CNC directly interacts with the interface without deforming it. As a


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result, only surface interactions occur between the CH of the CNC and the alkyl chain of hexadecane. This experiment shows clearly that rigid nanoparticles can be densely adsorbed at the oil/water interface without deforming it at the nanoscale.

Conclusion The particle structure at the oil/water interface of CNC-stabilized Pickering emulsions was investigated according to CNC concentration, with surface charges in presence of ionic strength, or without charges. It appeared that the drop size and coverage is influenced by the structure of the CNCs. The thickness of the interfacial layer calculated from the SANS measurements revealed a 7 nm monolayer, irrespective of the amount of CNCs. This was interpreted as a densification of the interface that might correspond to a local orientation/alignment of the CNCs. More aggregated neutral CNCs led to a more porous and heterogeneous surface, 18 nm thick, due to densification of the larger aggregates and resulting in a more rigid armor. Assuming that the less hydrophilic (2 0 0) crystalline plane is the face of the crystal in contact with oil, the diagonal dimension of the elementary unit given by Xray diffraction should then reach a value of approximately 7 nm, the same as the one found for a monolayer adsorbed at an interface. It is then postulated that the nanocrystals align along that (2 0 0) plane as a monolayer without deforming the oil interface.

Acknowledgements

This work is a contribution to the Labex Serenade (n° ANR-11-LABX-0064) funded by the «Investissements d’Avenir » French Government program of the French National Research Agency (ANR) through the A*MIDEX project (n° ANR-11-IDEX-0001-02). The authors acknowledge Joelle Davy for SEM visualization, Nicolas Stephan for SEM assistance (IMN, Nantes, France) and Emilie Perrin for the excellent technical support.


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References 1. Fratzl, P.; Weinkamer, R., Prog. Mater. Sci. 2007, 52, (8), 1263-1334. 2. Klemm, D.; Kramer, F.; Moritz, S.; Lindstrom, T.; Ankerfors, M.; Gray, D.; Dorris, A., Angew. Chem. Int. Ed. 2011, 50, (24), 5438-5466. 3. Lagerwall, J. P. F.; Schutz, C.; Salajkova, M.; Noh, J.; Park, J. H.; Scalia, G.; Bergstrom, L., 2014, 6, E80. 4. Ramsden, W., Proc. Roy. Soc. 1903, 72, 156. 5. Pickering, S. U., J. Chem. Soc. 1907, 91, 2001-2021. 6. Binks, B. P., Curr. Opin. Colloid Interface Sci. 2002, 7, (1-2), 21-41. 7. Leal-Calderon, F.; Schmitt, V., Curr. Opin. Colloid Interface Sci. 2008, 13, (4), 217-227. 8. Arditty, S.; Schmitt, V.; Giermanska-Kahn, J.; Leal-Calderon, F., J. Colloid Interface Sci. 2004, 275, (2), 659-664. 9. Arditty, S.; Whitby, C. P.; Binks, B. P.; Schmitt, V.; Leal-Calderon, F., Eur. Phys. J. E 2003, 11, (3), 273-281. 10. Kalashnikova, I.; Bizot, H.; Bertoncini, P.; Cathala, B.; Capron, I., Soft Matter 2013, 9, 952-959. 11. Loudet, J. C.; Alsayed, A. M.; Zhang, J.; Yodh, A. G., Phys. Rev. Lett. 2005, 94, (1). 12. Loudet, J. C.; Yodh, A. G.; Pouligny, B., Phys. Rev. Lett. 2006, 97, (018304). 13. Perrin, E.; Bizot, H.; Cathala, B.; Capron, I., Biomacromolecules 2014, 3766-3771. 14. Madivala, B.; Vandebril, S.; Fransaer, J.; Vermant, J., Soft Matter 2009, 5, (8), 1717-1727. 15. Luu, X.-C.; Striolo, A., J. Phys. Chem. B 2014, 118, (47), 13737-13743. 16. Klemm, D.; Heublein, B.; Fink, H. P.; Bohn, A., Angew. Chem. Int. Ed. 2005, 44, (22), 33583393. 17. Revol, J. F.; Bradford, H.; Giasson, J.; Marchessault, R. H.; Gray, D. G., Int. J. Biol. Macromol. 1992, 14, (3), 170-172. 18. Elazzouzi-Hafraoui, S.; Nishiyama, Y.; Putaux, J. L.; Heux, L.; Dubreuil, F.; Rochas, C., Biomacromolecules 2008, 9, (1), 57-65. 19. Cherhal, F.; Cathala, B.; Capron, I., Nord. Pulp Pap. Res. J. 2015, 30, (2), 126-131. 20. Cherhal, F.; Cousin, F.; Capron, I., Langmuir 2015, 31, (20), 5596-5602. 21. Araki, J., Soft Matter 2013, 9, (16), 4125-4141. 22. Kalashnikova, I.; Bizot, H.; Cathala, B.; Capron, I., Langmuir 2011, 27, (12), 7471-7479. 23. Kalashnikova, I.; Bizot, H.; Cathala, B.; Capron, I., Biomacromolecules 2012, 13, (1), 267-275. 24. Destribats, M.; Faure, B.; Birot, M.; Babot, O.; Schmitt, V.; Backov, R., Adv. Funct. Mater. 2012, 22, (12), 2642-2654. 25. Taisne, L.; Walstra, P.; Cabane, B., J. Colloid Interface Sci. 1996, 184, (2), 378-390. 26. 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, (48), 6455-6462. 27. Jean, B.; Dubreuil, F.; Heux, L.; Cousin, F., 2008, 24, (7), 3452-3458. 28. Jestin, J.; Simon, S.; Zupancic, L.; Barre, L., Langmuir 2007, 23, (21), 10471-10478. 29. Cotton, J. P., In Neutron, X-ray and Light Scattering. Lindner, P., Zemb, T., Eds.; North Holland: New York ed.; 1991. 30. Nishiyama, Y.; Sugiyama, J.; Chanzy, H.; Langan, P., J. Am. Chem. Soc. 2003, 125, (47), 1430014306. 31. Fink, H. P.; Hofmann, D.; Philipp, B., Cellulose 1995, 2, (1), 51-70. 32. Mazeau, K., Carbohydr. Polym. 2011, 84, (1), 524-532. 33. Shimon, L. J. W.; Pages, S.; Belaich, A.; Belaich, J. P.; Bayer, E. A.; Lamed, R.; Shoham, Y.; Frolow, F., Acta Crystallogr. Sect. D-Biol. Crystallogr. 2000, 56, 1560-1568. 34. Lehtio, J.; Sugiyama, J.; Gustavsson, M.; Fransson, L.; Linder, M.; Teeri, T. T., Proc. Natl. Acad. Sci. U. S. A. 2003, 100, (2), 484-489. 22 ACS Paragon Plus Environment

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TOC

CNC in water h = 7 nm

h = 18 nm

Smooth oil/water interface

Sulfated CNC

Desulfated CNC


Structural Description of the Interface of Pickering Emulsions

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