Effect of Counterion Choice on the Stability of Cellulose Nanocrystal

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Materials and Interfaces

The Effect of Counterion Choice on the Stability of Cellulose Nanocrystal Pickering Emulsions Lingli Liu, Zhen Hu, Xiaofeng Sui, Jing Guo, Emily D. Cranston, and Zhiping Mao Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.8b01001 • Publication Date (Web): 08 May 2018 Downloaded from http://pubs.acs.org on May 8, 2018

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Industrial & Engineering Chemistry Research

The Effect of Counterion Choice on the Stability of Cellulose Nanocrystal Pickering Emulsions Lingli Liu,†,‡ Zhen Hu‡, Xiaofeng Sui,† Jing Guo,§ Emily D. Cranston,*,‡ and Zhiping Mao*,†

†

Key Lab of Science and Technology of Eco-textile, Ministry of Education, College of

Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, China. ‡

Department of Chemical Engineering, McMaster University, Hamilton, ON L8S 4L7, Canada

§

Dow Food, Pharma and Medical. Dow Chemical Company, Midland Michigan 48674, USA

KEYWORDS: cellulose nanocrystals, counterions, oil type, Pickering emulsions, stability to coalescence, creaming, corn oil, hexadecane, pH, ionic strength

ABSTRACT: Cellulose nanocrystals (CNCs) with three different counterions (H+, Na+, K+) were used to prepare oil-in-water Pickering emulsions with and without salt; their stability to coalescence and resistance to creaming was tested using two oil types (high-polarity corn oil and super-low polarity hexadecane). Without salt, only acid-form CNCs could stabilize corn oil emulsions, whereas the salt-form Na+-CNCs and K+-CNCs failed. None of the CNCs could stabilize hexadecane/water emulsions due to the lack of oil-CNC interactions and strong repulsion between CNCs. However, adding salt masked the differences between the CNC types and all CNCs could stabilize both corn oil and hexadecane emulsions. Unfortunately, when salt was added, extensive creaming occurred owing to the water-phase density increase and droplet 1

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aggregation. The effect of salt concentration and neutralization of CNCs by different methods was also investigated. This work highlights the complex nature of CNC interactions with salts, oils, acids/bases and other additives, which is relevant for envisioned formulated products for food, cosmetic and pharmaceutical applications, and reveals that the choice of CNC counterion does influence emulsion performance. INTRODUCTION Pickering emulsions are stabilized by anchoring colloidal particles with intermediate wettability at the interface of two immiscible liquids. Despite their discovery in the early 1900s, research gained momentum in the fields of food, cosmetics, pharmacy, farming, etc. in the 1970s.1,2 In addition to retaining the general properties of traditional surfactant-stabilized emulsions, Pickering emulsions have colloidal particles that are irreversibly attached to the interface,3,4 which implies that droplet coalescence and Ostwald ripening are strongly inhibited.5 The superior stability of Pickering emulsions makes them attractive for most emulsion applications with the further advantage of often being prepared with food-grade or functional particles that can impart new properties to the system. The two main types of stabilizing particles are (1) inorganic colloidal particles, such as silica,6 calcium carbonate,7 clays8 and titanium dioxide,9 and (2) organic colloidal particles, such as carbon nanotubes,10 block copolymer micelles,11 and nanocellulose,12–16 to name just a few. Cellulose nanocrystals (CNCs) are an emerging nanomaterial17 suitable for use in Pickering emulsions. CNCs are extracted from natural resources through a low carbon footprint production route, are non-toxic18 and biodegradable, and have been widely demonstrated to enhance various materials (e.g., emulsions,19,20 hydrogels,21,22 aerogels,23,24 composites,25,26 adhesives,27 and films/coatings28,29). CNCs are most commonly isolated through acid hydrolysis of cotton or

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wood.30–34 For example, sulfuric acid can be used to selectively hydrolyze less ordered cellulose, producing highly crystalline rod-like cellulose nanoparticles with anionic sulfate half esters grafted onto the surface.35 Generally, CNCs from plants have a high aspect ratio with lengths of 100-300 nm, and cross sections of 5-10 nm.17 At the end of the hydrolysis reaction, CNCs are in what is called the protonated acid-form (H+-CNCs)17 and if dried, CNCs in this form are non-redispersible in water due to enhanced hydrogen bonding compared to their corresponding neutralized salt-form (i.e., Na+-CNCs, K+-CNCs, etc.).36,37 Based on storage and transport requirements, most of the industrially produced CNC materials currently available are distributed as dry powders in their salt-form produced by titrating H+-CNCs with strong base (normally NaOH) to a pH of 5 ~ 7 prior to spray or freeze drying. As such, the development of most commercial products will need to begin with dried Na+-CNCs (that are straightforward to disperse in water37 or can be added directly to polymer melts, for example); however, we believe that for some intended applications, the counterion affects the CNC behavior. Specifically, adding dried Na+-CNCs directly to emulsion formulations may not be the ideal route. Capron and co-workers pioneered the use of CNCs as Pickering emulsifiers in 2011 when they reported the first oil-in-water (O/W) Pickering emulsion stabilized by cellulose nanocrystals made from bacterial cellulose.38 CNCs were obtained from hydrochloric acid hydrolyzed bacterial cellulose, which gave uncharged CNCs thereby minimizing particle-particle repulsion and facilitating particle partitioning at the non-polar hexadecane-water interface.38 Adding salt to charged CNCs to screen electrostatic repulsion similarly improved the stabilization abilities of CNCs.39 To better understand the CNC stabilization mechanism, they used wide-angle X-ray scattering to investigate the amphiphilic surface character of CNCs from two cellulose origins

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and concluded that the CNCs’ amphiphilic character can be ascribed to the crystalline organization at the elementary level.39 The amphiphilic nature of CNCs results from the orientation of the crystalline cellulose planes with the equatorial plane edge exposing hydrophilic hydroxyl moieties and the axial edge (200 plane) exposing hydrophobic -CH moieties.39 Capron’s research team further studied CNC emulsion performance as a function of CNC type, oil/water ratio, ionic strength, and CNC surface modification, to produce new emulsions, such as high internal phase emulsions (HIPEs), water-in-water emulsions and oil-in-water-in-oil double emulsions.13,40–43 Other researchers explored the potential of CNC-stabilized emulsions by modifying CNCs with stimuli responsive polymers44,45 and particles20 as described in a recent review.46 Based on these developments, the practical application of CNC Pickering emulsions in areas such as pharmaceuticals, cosmetics, food and oil recovery seems closer than ever. Only a handful of previous reports have studied how CNC counterions affect material behavior, and most papers omit to even mention what “form” the CNCs used are in, i.e., acid or salt-form. In one early example, Gray and coworkers36,47 reported a comprehensive investigation on the effect of CNC counterions (inorganic series Na+, K+ and Cs+, and organic series H+, NH(Et)3+, NH4+, etc.) on suspension properties. CNCs are lyotropic liquid crystals and the counterions on the sulfate half ester groups were found to significantly affect CNC self-assembly and phase separation. By changing only the counterion, while carefully keeping all the other conditions the same, they found that the counterion properties, like hydration number, dissociation constant, ion size and hydrophilic-hydrophobic balance, played a vital role in the isotropic and anisotropic phase equilibrium because they changed the interparticle forces in suspension.36,47

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We have also shown previously that the use of co-stabilizers that adsorb to CNCs, such as cationic surfactants (cetyl trimethylammonium bromide, didodecyldimethylammonium bromide – which is essentially a counterion exchange from H+ or Na+ to a bulky amphiphilic alkylammonium cation),48–50 and non-ionic polysaccharides (methyl cellulose, hydroxyethyl cellulose),12,19 can greatly improve the stabilizing abilities of CNCs. Pickering emulsions stabilized by CNCs and co-stabilizers are resistant to coalescence and have smaller oil droplets because the CNC surface charge is screened and the amphiphilicity is increased.12 Most recently, we recognized the effect of counterion type on surfactant adsorption to CNCs, which was investigated using colloid probe atomic force microscopy (AFM), light scattering and zeta potential, and indicated that greater adsorption (but less ordered surfactant structures) was promoted with Na+-CNCs compared to H+-CNCs.50 This trend in surfactant association/self-assembly followed the Hofmeister series which predicts the ability for ions to strengthen hydrophobic interactions.51 It has also been highlighted in the literature that ion interactions with CNCs are complex and can lead to gelation (especially if multivalent ions are used).52 To the best of our knowledge, no previous work has studied the influence of CNC counterion type on the ability of CNCs to stabilize Pickering emulsions. Herein, Pickering emulsions stabilized with H+-CNCs, Na+-CNCs, and K+-CNCs were prepared by emulsifying corn oil or hexadecane-in-water, with or without salt. The effect of the counterion choice on the final Pickering emulsion properties is described, including oil droplets size, oil leakage, creaming and shelf-life stability. We demonstrate that stable emulsions without oil leakage (which is critically important for most commercial applications) can be produced, yet an increase in droplet size or droplet aggregation often occurs. Although emulsion degradation mechanisms for small molecule surfactants have mostly focused on coalescence, flocculation and

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Ostwald ripening,5 in the case of Pickering emulsions, the particles are irreversibly anchored at the interface such that no stabilizer dissolution, diffusion or redepositing can occur. As such, Ostwald ripening is negligible and the particles at the interface serve as barriers to prevent the oil droplets from coming into physical contact with each other. Additionally, lowering the interfacial tension and increasing the emulsion viscosity enhances emulsion stability, as demonstrated here.

EXPERIMENTAL SECTION Materials. Corn oil, hexadecane, sodium chloride, sodium hydroxide, potassium hydroxide, triethylamine (TEA) and Oil Red O were purchased from Sigma-Aldrich (Oakville, ON, Canada). Sulfuric acid was purchased from Caledon Laboratory Chemicals (Georgetown, ON, Canada). Whatman cotton ashless filter aid was purchased from GE Healthcare Canada. All chemicals were used as received, unless otherwise specified. All water used was purified type I water with a resistivity of 18.2 MΩ·cm (Barnstead NANOpure DIamond system, ThermoScientific, Asheville, NC). Preparation of Acid, Sodium and Potassium Form Cellulose Nanocrystals. CNCs were prepared by sulfuric acid hydrolysis of cotton, as previously described.17 In brief, under continuous mechanical stirring, 40 g cotton Whatman ashless filter aid was treated in 700 mL sulfuric acid (64 wt.%) at 45℃ for 45 min. Then the hydrolysis procedure was quenched by adding a 10-fold volume of cold water. Multiple rinsing, centrifugation (6000 rpm in 10 min increments), and extensive dialysis against purified water were performed to remove excess acid and degraded sugars until the pH of the water outside the dialysis tubes remained at 5 ~ 6. The suspension was then probe sonicated (Sonifier 450, Branson Ultrasonics, Danbury, CT) for 45 min at 60% output in an ice bath. After that, the suspension was filtered through Whatman glass

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fiber filter paper (grade GF/B filter for liquid scintillation, with nominal particle retention of 2.7 µm) to remove contaminants from the sonicator probe and large aggregates. Sodium hydroxide (1 mM) was added to neutralize the CNC suspensions to pH 7, which was necessary for the lyophilization, storage and resuspension by sonication.37 The average sulfate half ester content determined by conductometric titration31 was 237 mmol/kg of CNCs, corresponding to 0.76% S and 0.37 charges/nm2. The average dimensions measured by AFM were 128 ± 50 nm in length and 7 ± 5 nm in cross section which is consistent with previous reports.17 The CNCs have a crystallinity index of 93% measured by X-ray diffraction and are in the cellulose I crystal form.17 As discussed by Abitbol et al.53 the sulfur contents measured by both conductometric titration and elemental analysis generally show good statistical accordance and we believe that titrations offer more precision to the measurement. Sodium-form CNC (Na+-CNC) suspensions were prepared by probe sonicating freeze-dried sodium-form CNCs in purified water at ca. 0.5 wt.% in an ice bath three times (5 min each time at 60% output), then filtered through Whatman glass fiber filter paper. Na+-CNCs are known to be easily redispersible in water37 and so after ensuring proper dispersion, Na+-CNCs were ion exchanged appropriately. Acid-form CNCs (H+-CNCs) were collected by passing the Na+-CNC suspension through a Dowex Marathon C hydrogen form ion-exchange resin column, a strong acid cation exchange resin. The resin removes the Na+ from sodium-form CNCs and exchanges it for H+ without changing the sulfate half ester content.53 Overall, the ion-exchange process converts Na+-CNC suspensions into H+-CNC suspensions and lowers the pH from 7 to about 3. Potassium form CNCs (K+-CNCs) were obtained by adding 1 mM potassium hydroxide to the H+-CNC suspension until it reached the pH 6.6 which is the same as Na+-CNCs. This highlights that the CNC counterion is in fact a “choice” and that counterions can be exchanged in a fairly

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straightforward manner. All the CNC types were diluted to 0.25 wt.% with purified water before being used for the preparation of Pickering emulsions. Preparation of the Pickering Emulsions. The corn oil-in-water Pickering emulsions were prepared with corn oil and 0.25 wt.% CNC suspension at an oil/aqueous phase volume ratio of 1/4. Briefly, 2 mL corn oil was first vortex-mixed with 8 mL CNC suspension (0.25 wt.%), then emulsified using the probe sonicator for 3 min, in an ice bath at an intensity level 6 and 50% pulses. The hexadecane-in-water Pickering emulsions were prepared with the same recipe, except the oil phase was hexadecane which was dyed with Oil Red O (0.5 mg/mL to the hexadecane volume) prior to use. The Pickering emulsions using only CNCs were denoted as H+-CNC, Na+-CNC, and K+-CNC Pickering emulsions. For some samples, 50 mM NaCl was added into the aqueous phase before mixing with the oil phase, which were specified with the NaCl suffix in the sample name. Dynamic Light Scattering (DLS). To measure the apparent particle size, i.e., the hydrodynamic diameter, of CNCs in suspension, a Malvern Zetasizer Nano particle analyzer was used at 20℃. “Apparent particle size” is written because the DLS measurement assumes spherical particles, while CNCs are rod-shaped making the measurement relative, at best. All the samples were diluted with purified water to the concentration of 0.025 wt.% before DLS measurements. (While NaCl is often added to collapse the double layer and obtain accurate DLS values for charged spherical particles, we hesitate to add salt to our CNC suspensions because we do not want to induce any aggregation54 and do not want to risk counterions exchanging for Na+ ions – since we only take DLS values of CNCs to be relative and “apparent” this omission seems reasonable.) The results shown here are an average of three measurements, and error bars represent the standard deviation.

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Zeta-Potential. A zeta-potential ZetaPlus analyzer (Brookhaven, USA) was used to measure the electrophoretic mobility and zeta-potential of CNC suspensions with different counterions. All the CNC samples were diluted with purified water to 0.25 wt.% before measuring (no salt added). The results shown here are an average of three measurements, and error bars represent the standard deviation. Malvern Mastersizer. To measure the Pickering emulsion oil droplet diameter, a Malvern Mastersizer 2000G instrument (laser diffraction particle sizing) was used with a 633 nm Helium neon laser. The average droplet diameter was calculated from the three replicate measurements of the volume mean diameter (D4/3) based on the Mie scattering measurement principle. According to the British Standard BS2955:1993,55 it is mathematically defined as shown in Equation 1. 4

D4 / 3 =

∑ Di N i 3 ∑ Di N i

Equation 1

where Di is the droplet diameter of droplet fraction i, Ni is the droplets number of size Di. The refractive index of the oil phase used in the Malvern Mastersizer software during measurements was 1.472 for corn oil and 1.434 for hexadecane. The result shown here are an average of three measurements, and error bars represent the standard deviation. Optical Microscopy (OM). An Axiovert 100M microscope (Zeiss, Gottingen, Germany) was used to visualize the Pickering emulsions. The emulsions were 100-fold diluted before being dropped onto a glass slide, and covered by a coverslip glass. The irregular or elliptical (grey-ish) areas in some of the OM images below were identified as partially leaked oil phase. Surface and Interfacial Tension. The surface tension of CNC suspensions with different counterions and their interfacial tension at the oil-water interface as a function of CNC concentration were measured by a Krüss Drop Shape Analysis System DSA10 (Hamburg, 9



Germany) via the pendant drop method. For the interfacial tension measurement, a glass box of 5.0 (L) × 5.0 (W) × 5.0 (H) cm was half-filled with oil sample. A 1 mL tuberculin syringe with its stainless steel needle immersed in the oil phase was used to control the droplet forming. The data were collected after 20 min ageing which was believed to be the equilibrium results. The results shown here are an average of three measurements, and error bars represent the standard deviation. Contact Angle Measurements at the Water-CNC Interface, Oil-CNC Interface and Oil-CNC-Water Interface. The above Krüss Drop Shape Analysis System DSA10 was also used to measure static contact angle of CNC films (with H+, NH(Et)3+, Na+ and K+ counterions) toward three different systems (water/film/gas (W/F/G), oil/film/gas (O/F/G) and water/film/oil (W/F/O)). All samples were measured at room temperature (25 ± 2℃). 2 wt.% CNC suspension was spin-coated (G3P Spincoat, Specialty Coating Systems Inc. Indianapolis, USA) on polished Si wafers ((MEMC Electronic Materials Sdn Bhd, Petaling Jaya, Malaysia) under N2 gas at 3000 rpm for 60 s, each wafer was spin-coated three times to ensure full surface coverage, followed by drying at 80℃ overnight. Prior to coating, the Si wafers were cleaned in a piranha solution (1:3 vol:vol hydrogen peroxide to concentrated sulfuric acid) for half an hour, then rinsed with running Mill-Q water and dried in N2 gas. The contact angle at W/F/G or O/F/G systems was measured with a 3 µL sessile drop of purified water or oil placed on the CNC film under air. The three-phase contact angle at W/F/O system was measured with a 3 µL sessile drop of purified water placed on the CNC film under oil (corn oil, or hexadecane). Specifically for the three-phase contact angle measurement, the CNC spin-coated Si wafer was immersed in a glass box of 5.0 (L) × 5.0 (W) × 5.0 (H) cm partly filled with oil, followed by adding 3 µL water droplet. Measurements were conducted on three drops, then averaged. Each measurement was

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done on a new spot on the film. The droplet images were recorded and analyzed using ImageJ software. The error bars represent the standard deviation.

RESULTS AND DISCUSSION Physical Properties of CNC Starting Materials and Oil Types Used in Pickering Emulsions. To understand how the counterions on the sulfate half ester groups on the CNCs affect O/W emulsion performance, the physical properties of the starting CNCs and oils were characterized (Table 1). CNC suspensions with H+, Na+ and K+ counterions were characterized by apparent particle size, zeta-potential, pH and surface tension. Except for pH, the other physical properties did not vary significantly with counterion type and the CNCs were all well-dispersed and colloidally stable in water. While CNCs are amphiphilic39 and have been found to have intermediate wettability,48 the surface tension values in Table 1 show that they are not strictly surface active by definition, as the reduction in surface tension is minimal. Two distinct oil types, corn oil and hexadecane, were chosen for the oil phase in the CNC-stabilized Pickering emulsions. The molecular weight (Mw), density, viscosity, polarity and water solubility of corn oil are all significantly larger than for hexadecane, especially the latter three properties. Corn oil also has a higher surface tension than hexadecane56 indicating that corn oil molecules exhibit stronger cohesion (and lower adhesion with air) than hexadecane. The interfacial tension of corn oil/water, however, is significantly lower than for hexadecane/water, which can be explained by the surface active compounds in corn oil, such as triglycerides, diglycerides, monoglycerides and free fatty acids.57 These compounds can diffuse rapidly to stabilize the O/W interface and lower the difference between oil molecule cohesion

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and oil/water adhesion. In contrast, non-polar hexadecane is a pure single component oil composed entirely of hydrocarbon chains and does not contain any surface active compounds.57 Table 1. Physical properties of CNCs in suspension with different counterions and the oil types used to prepare Pickering emulsions. Electrophoretic CNC Apparent particle Zeta-potential Surface tension mobility pH suspension size (nm) (mV) (mN/m) (×10-8 m2s-1V-1) H+-CNCs

78 ± 33

-42 ± 1

-3.31 ± 0.05

71 ± 0.6

3.6

Na+-CNCs

78 ± 31

-41 ± 1

-3.29 ± 0.03

72 ± 0.6

6.6

K+-CNCs

79 ± 30

-42 ± 1

-3.30 ± 0.08

71 ± 0.3

6.6

Oil type

Mw (g/mol)58

Corn oil

800 ~ 900

Hexadecane

226

Density Viscosity Water (kg/m3, (mPa·s, Polarity59 solubility60 (mg/L) 25℃) 20℃)

Surface tension (mN/m, 25℃)

Interfacial tension (mN/m, 25℃, O/W)

920

88 ± 3

3.0 ~ 3.2

6.4

34.0 ± 0.5

23.5 ± 0.5

770

4±1

0

2.1×10-5

27.5 ± 0.5

53.5 ± 0.3

Effect of Counterion Choice on CNC-stabilized Pickering Emulsion Properties. To provide insight into the stability of CNC-stabilized O/W Pickering emulsions with different counterions, photographs, Mastersizer measurements and optical microscopy were used to monitor the emulsion performance and changes over a period of 720 h, including oil leakage, creaming, droplet size, and droplet morphology. Figure 1 shows the photographs of O/W Pickering emulsions stabilized by H+-CNCs, Na+-CNCs, and K+-CNCs with and without 50 mM NaCl added, before and after 720 h storage. Corn oil is visibly yellow and hexadecane is dyed by Oil Red O (that appears pink) prior to preparing emulsions; this helps to facilitate observation of oil leakage which appears as the upper phase. Figure 2 quantifies the emulsion resistance to oil leakage and Figure 3 the resistance to creaming (based on the photographs in Figure 1). Intermediate ageing times are shown in more detail in Figure 4, which tracks the change of

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emulsion droplet size by Mastersizer. For all systems studied, emulsions could be prepared but it is the behavior over time that offers new insight into CNC Pickering emulsion stability. Without added salt, the CNC Pickering emulsions with different counterions showed different behavior (Figure 1a – 1d). When highly polar corn oil with high water solubility was used as the oil phase, stable Pickering emulsions were obtained only with H+-CNCs; Na+-CNC and K+-CNC emulsions showed significant oil leakage after 720 h (Figure 1b). The leaked corn oil volume percentages were 25% and 75% for Na+-CNCs and K+-CNCs, respectively (Figure 2a and 2b). When the oil phase was changed to hexadecane, which has a very low polarity and water solubility, none of the CNC types gave stable emulsions and hexadecane leakage was observed after 720 h (Figure 1c and 1d). The least oil leakage occurred for H+-CNC emulsions (15%), corresponding to the lightest pink color upper phase. Na+-CNC and K+-CNC emulsions lost 30% and 35% of their original oil volume, respectively. Due to the fairly hydrophilic and polar nature of CNCs, polar corn oil (with its lower interfacial tension) is easier to stabilize compared with hexadecane and in all cases, H+-CNCs stabilize better than Na+-CNCs which are in turn better than K+-CNCs. Although the three types of CNCs with different counterions had the same surface tension, we believe the surface active compounds in corn oil have the strongest interactions (e.g., hydrogen bonding and van der Waals) with H+-CNCs, thus contributing to the successful formation of corn oil-in-water Pickering emulsions. While H+-CNC emulsions with corn oil did not cream or phase separate in the absence of salt (Figure 1b), hexadecane emulsions broke and phase separated (Figure 1d) after 720 h. More specifically, the emulsion phases of the CNC-stabilized hexadecane-in-water emulsions all decreased from 100% to around 30% after storage (Figure 3a and 3b). The less favorable

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interaction between CNCs and the hexadecane-water interface, and the significant density difference between the phases are the factors primarily responsible for this behavior.13 In the presence of added salt, all of the CNC Pickering emulsions were stable, and no oil leakage occurred for either corn oil or hexadecane (Figure 1e – 1h and Figure 2c and 2d). No noticeable difference was observed between emulsions prepared with CNCs with different counterions or with different oil types. However, the addition of salt caused severe creaming (Figure 1f and 1h). Creaming is the migration of the dispersed phase in an emulsion and is attributed to the impact of buoyancy. The emulsion droplets flow upwards because the density of the continuous phase (water) is higher than the dispersed phase (oil), while the emulsion viscosity is not high enough to prevent the mobility of droplets.61,62 This effect is exacerbated for large oil droplets but also for aggregated small droplets. While creaming is undesirable, it does not represent a broken emulsion or droplet coalescence – commercial products often contain rheological modifiers to restrict oil droplets from aggregating and rising to the surface.38,63 After 720 h of storage at room temperature for emulsions with salt, the emulsion phase decreased in volume from 100% to 37 – 47% (Figure 3c and 3d). This results from both the density difference between phases and the screening of electrostatic repulsion between droplets which facilitates droplet aggregation.64 Adding 50 mM NaCl increases the density of the water phase from 1.00000 × 103 kg/m3 to 1.00292 × 103 kg/m3, creating a slightly larger density difference between water and corn oil/hexadecane which increases creaming velocity.65 Despite this creaming, and in accordance with previous findings,12,39 all CNCs with added salt effectively stabilized emulsion droplets in water (Figure 2c and 2d). The droplet resistance to coalescence is highlighted in the droplet size evaluation presented in the following section.

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Figure 1. Photographs of corn oil-in-water Pickering emulsions (oil:water volume ratio 1:4) stabilized by 0.25 wt.% CNCs (H+, Na+, or K+ counterions) right after emulsification (a), and after storage at room temperature for 720 h (b). Hexadecane-in-water Pickering emulsions (volume ratio 1:4) stabilized by 0.25 wt.% CNCs (H+, Na+, or K+ counterions) right after emulsification (c), and after storage at room temperature for 720 h (d). Corn oil-in-water Pickering emulsions (volume ratio 1:4) stabilized by 0.25 wt.% CNCs (H+, Na+, or K+ counterions) in the presence of 50 mM NaCl right after emulsification (e), and after storage at room temperature for 720 h (f). Hexadecane-in-water Pickering emulsions (volume ratio 1:4) stabilized by 0.25 wt.% CNCs (H+, Na+, or K+ counterions) in the presence of 50 mM NaCl right after emulsification (g), and after storage at room temperature for 720 h (h).

Figure 2. Resistance to oil leakage: The volume percent of oil retention in Pickering emulsions stabilized by 0.25 wt.% CNCs (H+, Na+, or K+ counterion) right after emulsification (a), and after storage at room temperature for 720 h (b). The volume percent of oil retention in Pickering 15



emulsions stabilized by 0.25 wt.% CNCs (H+, Na+, or K+) in the presence of 50 mM NaCl right after emulsification (c), and after storage at room temperature for 720 h (d). The volume percent of oil retention indicates the volume percent of oil that remained encapsulated in the emulsion droplets.

Figure 3. Resistance to creaming: the volume percent of emulsion phase in Pickering emulsions stabilized by only 0.25 wt.% CNCs (H+, Na+, or K+) right after emulsification (a), and after storage at room temperature for 720 h (b). The volume percent of emulsion phase in Pickering emulsions stabilized by 0.25 wt.% CNCs (H+, Na+, or K+) in the presence of 50 mM NaCl right after emulsification (c), and after storage at room temperature for 720 h (d).

Complementary to the observations in Figure 1, the average emulsion droplet diameters (D4/3 described by Equation 1) measured by Mastersizer are shown in Figure 4. Without salt, all droplet sizes increased over time and plateaued around 240 h. The droplet size was more dependent on counterion type with corn oil than with hexadecane, and the smallest droplets were seen with H+-CNCs and corn oil. More specifically, in the absence of 50 mM salt, the droplet diameters were 3 µm, 11 µm, and 7 µm for corn oil-in-water emulsions with H+-CNCs, Na+-CNCs and K+-CNCs, respectively, immediately after emulsification. After 240 h, H+-CNCs emulsion droplet diameter increased to 6 µm and no further growth was seen with longer storage times. However, the droplet diameter of corn oil emulsions with Na+-CNCs and K+-CNCs both gradually increased to 23 µm over 720 h. The situation was quite similar for H+-CNC, Na+-CNC

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and K+-CNC Pickering emulsions with hexadecane; droplets diameters increased from ca. 5 µm to 25 µm over 720 h (Figure 4a). In contrast, with added salt, all emulsion droplets were smaller, i.e., 2–5 µm, and changed minimally over time irrespective of counterion and oil type (Figure 4b). This is attributed to the ability of salt to screen electrostatic repulsion which allows CNCs to pack more densely at the droplet interface.4,54 We additionally note that during the Mastersizer measurements, the emulsion samples were continually circulated through hydrophobic silicone rubber tubing and while stable emulsion droplets without oil leakage are not expected to stick to the tubing, unstable emulsions with oil leakage may stick to the lining before reaching the measurement cell. This may reduce the number of droplets measured and slightly skew the droplet sizing. As such, optical microscopy images are shown in Figure 5 and Figure 6; optical microscopy is suggested to be a more reliable technique to understand how emulsion droplets behave over time.

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Figure 4. Average droplets diameter of 1:4 O/W emulsions without salt stabilized by 0.25 wt.% CNCs (H+, Na+, or K+ counterions) over 720 h (a), 1:4 O/W emulsions with salt stabilized by 0.25 wt.% CNCs (H+, Na+, or K+ counterions) in the presence of 50 mM NaCl over 720 h (b), which was measured by a Mastersizer. Each point is an average of three measurements, and error bars represent the standard deviation. Lines drawn between points are provided to guide the eye.

The effect of CNC counterion choice on the morphology of O/W CNC-stabilized Pickering emulsions with or without salt is shown in Figure 5 and Figure 6. As described above, only H+-CNCs gave a stable O/W emulsion for corn oil without salt. The average droplet size increased very slowly, which we infer was due to slight droplet coalescence rather than Ostwald ripening, because of the irreversible adsorption of CNCs at the interface.44 As expected, no trace of oil droplets was found in the optical micrographs, initially (Figure 5a) or after 720 h storage (Figure 5b). On the other hand, big emulsion droplets and sometimes ill-defined shapes of oil-only areas were observed in the optical micrographs for corn oil emulsions with both Na+-CNCs and K+-CNCs. It seems that the K+-CNC emulsions deteriorated more than Na+-CNC emulsions, for both corn oil (Figure 5f and 5j) and hexadecane (Figure 5h and 5l) after 720 h. Unfortunately, H+-CNCs alone did not work well to stabilize hexadecane either and in addition 18


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to severe oil leakage, the emulsion droplets themselves coalesced drastically after 720 h of storage (Figure 5d). These results demonstrate that the interaction between CNC counterions and oil strongly affects the emulsion properties, particularly when Pickering emulsions are prepared without salt.

Figure 5. Optical micrographs of 1:4 corn oil-in-water emulsions without salt stabilized by 0.25 wt.% CNCs (H+, Na+, or K+ counterions) right after emulsification (a, e, i) and after 720 h (b, f, j); 1:4 hexadecane-in-water emulsions stabilized by 0.25 wt.% CNCs (H+, Na+, or K+ counterions) right after emulsification (c, g, k) and after 720 h (d, h, l). All scale bars are 20 µm.

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Figure 6. Optical micrographs of 1:4 corn oil-in-water emulsions with salt stabilized by 0.25 wt.% CNCs (H+, Na+, or K+ counterions) in the presence of 50 mM NaCl right after emulsification (a, e, i) and after 720 h (b, f, j); 1:4 hexadecane-in-water emulsions stabilized by 0.25 wt.% CNCs (H+, Na+, or K+ counterions) in the presence of 50 mM NaCl right after emulsification (c, g, k) and after 720 h (d, h, l). All scale bars are 20 µm.

The droplet diameter analysis by Mastersizer and optical microscopy implies that the addition of salt “masks” the superior ability of H+-CNCs to stabilize corn oil-in-water emulsions compared to the salt-form CNCs and makes CNCs more “equivalent” emulsion stabilizers. By screening the negative surface charges on sulfated CNCs, the nanoparticles are more likely to partition at the oil-water interface, rather than be dispersed in the water phase, and the interfacial layer assembled can be denser.4 More CNCs at the interface results in better emulsion performance, such as smaller droplet diameters (more interfacial area) and more stable emulsion

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droplets (less droplet coalescence and oil leakage). This is supported by optical micrographs in Figure 6a – 6l in which all emulsion droplets with salt were found to be very small, around 2–3 µm immediately after emulsification and grew by less than 2 µm after 720 h storage. In addition, more emulsion droplet aggregation was generally seen in the Pickering emulsions in the presence of salt after 720 h storage because screening the surface charge of the droplets allowed droplets to come closer together but the CNC layer at interface still prohibited coalescence (this is exacerbated for hexadecane compared to corn oil because of the lower density and viscosity) – this droplet aggregation is correlated with the creaming phenomena observed in Figure 1. Even though the individual droplets remain below 5 µm, the aggregated drops are sufficiently large that gravitation forces overtake Brownian motion. Effect of Salt on CNC Pickering Emulsions. As discussed above for stabilizing corn oil-in-water Pickering emulsions without salt, H+-CNCs are more effective at stabilizing emulsions than Na+-CNCs and K+-CNCs, while the presence of salt obscures the distinction between CNC counterion types. Both Kalashnikova39 and Wen66 reported that CNC suspensions do not show visible aggregation below 100 mM of added salt (although nanoscale association and small aggregates are known to form in this salt range54) and that the emulsion volume remains constant after centrifugation in the presence of as little as 20 mM salt. In order to gain insight into the influence of salt on the CNC-stabilized corn oil emulsions, i.e., answer the question, “How much salt is needed to “mask” the effect of CNC counterions?”, 0, 5, 10, 20 and 50 mM salt were added to the CNC suspensions before mixing with the oil phase. (This was only done for corn oil since the behavior of corn oil and hexdecane emulsions with 50 mM added salt were indistinguishable in Figure 1, 2 and 3.) In all cases, the CNC suspensions appeared

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colloidally stable prior to emulsification. The emulsion properties are shown in Figure 7 and Table 2. The Pickering emulsions prepared from H+-CNCs with various salt concentrations all showed very stable performance. The small droplet size remained constant and no oil leakage occurred during 72 h storage at room temperature. Creaming only occurred for emulsions with 50 mM salt due to the imbalance of emulsion density and viscosity, as well as the emulsion droplet aggregation (Figure 7a and 7b, Table 2). However, Na+-CNC-stabilized emulsions behaved very differently. Stable emulsions could not be formed until the salt concentration surpassed 20 mM, which is in accordance with Capron’s previous reports.39,66 Extensive corn oil leakage coupled with increasing droplet sizes were observed for Na+-CNC emulsions with 0, 5 and 10 mM salt. Adding 50 mM salt also caused the emulsions to cream. As a result, in the corn oil/water system, the addition of 20 mM salt effectively “masks” the differences between CNC types (acid-form and salt-form) and is highlighted in Table 2 as the roughly optimized condition to avoid oil leakage and creaming.

Figure 7. Corn oil-in-water Pickering emulsions (1:4 by volume) stabilized by 0.25 wt.% H+-CNCs and NaCl with concentrations from 0 to 50 mM (left to right) right after emulsification (a) and after 72 h (b). Corn oil-in-water Pickering emulsions (1:4 by volume) stabilized by 0.25 22


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wt.% Na+-CNCs and NaCl with concentrations from 0 to 50 mM (left to right) right after emulsification (c) and after 72 h (d).

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Table 2. Average droplet diameter, oil leakage and creaming performance of CNC-stabilized corn oil-in-water emulsions (with H+, Na+ and K+ counterions) with varying salt concentration (0-50 mM). The highlighted row is a roughly optimized formulation that avoids oil leakage and creaming for both CNC types. Droplet diameter Droplet diameter Oil leakage Creaming NaCl (µm, 0 h) (µm, 72 h) concentration H+Na+H+Na+H+- Na+-CN H+Na+-CN (mM) CNCs CNCs CNCs CNCs CNCs Cs CNCs Cs 0 3 11 3 21 no yes no no 5 3 13 4 23 no yes no no 10 3 11 4 17 no yes no no 20 4 3 4 4 no no no no 50 2 3 2 3 no no yes yes Effect of pH on CNC Pickering Emulsions. In order to understand the stabilizing mechanism of H+-CNCs with corn oil and ensure it is not simply an effect of the acidic suspension pH, the effect of pH on the H+-CNC Pickering emulsion performance was investigated. As shown in Table 1, the only significant difference between H+-CNCs and the salt-form CNCs is the suspension pH. The H+-CNC suspension without any additives (which has a pH of 3.6) was used as the control. The pH was increased to 6.6 (which is the salt-form CNC pH) by two different routes: Route 1 - by first producing the emulsion with H+-CNCs and then adding inorganic alkali NaOH to the pre-prepared emulsion; and Route 2 - by adding the weak organic base triethylamine (TEA) to the H+-CNC suspension before mixing with corn oil. TEA was used instead of NaOH in Route 2 to avoid producing inorganic salt-form CNCs. We also consider that NH(Et)3+ produced in the neutralization reaction will interact with the CNC sulfate half ester groups but also may interact directly with the aforementioned surface active compounds in corn oil. Note that no salt was added in these emulsions and the contribution to the ionic strength from added NaOH and TEA is considered negligible. Figure 8a shows the photographs of H+-CNC-stabilized corn oil-in-water emulsions under the different pH conditions after 3 months of storage. The photographs do not indicate any

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deterioration in emulsion stability, nor did the emulsion droplet diameter change, as discussed further below. No oil leakage or creaming happened during ageing. The droplet diameter of the emulsions at different pH values exhibited the same trend over time, increasing from around 3 µm to 6 µm over a period of 720 h (30 days) (Figure 8b). The only noticeable difference between the three emulsions is the distribution of droplet diameters. The acidic emulsion (pH 3.6, Figure 8c) and the emulsion neutralized by NaOH after emulsification (pH 6.6, NaOH, Figure 8e) presented a narrow, unimodal droplet diameter distribution, which implies that the emulsions had similar sized droplets and a uniform size distribution. As such, as long as the H+-CNC Pickering emulsions were prepared before changing the pH, they can be neutralized later without it changing the emulsion properties significantly. That is, the CNCs stabilizing the emulsions were either partly or fully unsusceptible to ion exchange by strong base at the interface, or were already arranged at the interface in a stable configuration that could not be changed even when the counterion was exchanged – we suspect the latter is more likely. On the other hand, the droplet diameter distribution of H+-CNC-stabilized emulsions neutralized with TEA was wider but the emulsions exhibited similar appearance and droplet diameters compared to the acidic ones. Figure 8d shows that with TEA, however, the droplet size distribution switched from a skewed single peak to a bimodal distribution after ageing for 720 h, indicating that both large and small droplets existed in the emulsion. TEA is a weak base; it may be that not all H+ counterions are exchanged to NH(Et)3+, and as such the TEA neutralization only partly compromises the H+-CNCs’ ability to stabilize corn oil-in-water Pickering emulsion. Overall, the ability of CNCs to stabilize corn oil-in-water emulsions follows the order: H+-CNCs > TEA-neutralized CNCs > Na+-CNCs > K+-CNCs. This finding regarding the effect of pH on

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CNC-stabilized emulsions is believed to broaden the applicability of CNCs as emulsifiers, as long as the correct “order of operations” and addition of formulation components is followed (e.g., with acids/bases and salts).

Figure 8. The effect of pH on the performance of salt-free corn oil-in-water Pickering emulsions (volume ratio 1:4) stabilized by 0.25 wt.% H+-CNCs over 3 months: pH = 3.6 (left), triethylamine (TEA) added to the H+-CNCs suspension before emulsification to reach the pH = 6.6 (middle), and NaOH added to the Pickering emulsion after emulsification to reach pH = 6.6 (right) (a). The droplet diameter (b) and distribution of corn oil-in-water Pickering emulsions (volume ratio 1:4) stabilized by 0.25 wt.% H+-CNCs over 720 h, at pH 3.6 (c), pH 6.6 by TEA (d), pH 6.6 by NaOH (e), Each point is an average of three measurements, and error bars represent the standard deviation. Lines drawn between points are provided to guide the eye. CNC Counterion and Wettability Analysis. To further investigate why CNCs with H+ counterions stabilize corn oil-in-water Pickering emulsions better than Na+ and K+, the wettability of four CNC films (H+-CNCs, TEA-neutralized CNCs - called NH(Et)3+-CNCs below, Na+-CNCs, and K+-CNCs) with water, corn oil and hexadecane was studied (Figure 9). As 26


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mentioned above, Gray et al.36 and Beck et al.37 reported that freeze-dried H+-CNCs have stronger interparticle bonding than salt-form CNCs, owing to the extra hydrogen bonds generated by the existence of H+ counterions, which was supported by IR spectroscopy. In our study, the number and position of hydroxyl and sulfate half ester groups on the CNCs with four types of counterions are identical, and their intramolecular hydrogen bonding should be similar (i.e., within the nanoparticles) but intermolecular hydrogen bonding also appears stronger for our H+-CNCs. The CNCs may also differ slightly in how charged the sulfate half ester groups “appear” (i.e., the degree of dissociation) under the solution conditions employed and how the counterions interact with the surrounding water/oil molecules. From the cited studies and our new data, we propose that the presence of stronger interparticle binding between H+-CNCs as well as stronger H-bonding interactions with the surface active groups in corn oil may be responsible for more favorable interactions between H+-CNCs and corn oil compared to the salt-form CNCs. Figure 9 shows the wetting behavior of CNC films (with H+, NH(Et)3+, Na+, and K+ counterions) at W/F/G, O/F/G and W/F/O systems. It is apparent that the H+ and NH(Et)3+-CNC films had a higher water contact angle than Na+ and K+-CNC films, though all the contact angles decreased with time, reaching values lower than 5° after 10 min, due to absorption of water and evaporation (Figure 9a). The water contact angle testing supported that H+ and NH(Et)3+-CNCs have a lower wettability than Na+ and K+-CNCs, which is in accordance with previously reported work on H+ and Na+ CNCs.67,36,68 The wettability of corn oil and hexadecane on CNC films in air is depicted in Figure 9b; CNC films have much lower contact angles against hexadecane (0°) than corn oil (12° ~ 16°) due to the surface tension difference. Notably, the corn oil contact

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angles did not decrease significantly with time (due to their viscosity), and are lower than the water contact angles over the first 4 min, then higher as time continues. We can use the theoretical three-phase contact angle equation,69 Equation 2, to compare to the experimental three-phase contact angles shown in Figure 9c and 9d: cos θ3 =

γl1-g cos θ1 - γl2-g cos θ2 γl1-l2

Equation 2

where θ3 is the contact angle of liquid 1 in liquid 2, θ1 and θ2 are the contact angles of liquid 1 and liquid 2 in air, respectively, γl1-g and γl2-g are the liquid 1/gas and liquid 2/gas interfacial tension, also called surface tension, respectively, and γl1-l2 is the liquid 1/liquid 2 interfacial tension. Using the data from Figure 9a and 9b at 3 min (as an example) and Table 1, we can calculate the theoretical three-phase contact angle for water/H+-CNC/hexadecane as follows: If liquid 1 is water, its surface tension γl1-g is 72.8 mN/m, and the contact angle of water on H+-CNC film θ1 at 3 min is 21°. If liquid 2 is hexadecane, its surface tension γl2-g is 27.5 mN/m, and the contact angle of hexadecane on H+-CNC film θ2 at 3 min is 0°. The water/hexadecane interfacial tension γl1-l2 is 53.5 mN/m. As a result, θ3 = 41°. Likewise, the theoretical three-phase contact angle of water/NH(Et)3+-CNC/hexadecane at 3 min is the same at 41°, that of water/Na+-CNCs/hexadecane is 39°, and that of water/K+-CNC/hexadecane is 38°. These calculated values are very similar to each other and are relatively low (Bancroft’s rule indicates a three-phase contact angle close to 90° is ideal for Pickering stabilization). However, the same trend is seen experimentally (despite the experimental values being slightly lower at 32° for both H+-CNC and NH(Et)3+-CNC films, and 31° for both salt-form CNC films). When Equation 2 is applied to water/CNC/corn oil systems, however, the values of cos θ3 for H+-CNC, NH(Et)3+-CNC, Na+-CNC and K+-CNC films are 1.482, 1.482, 1.542 and 1.553, respectively, and θ3 is incalculable because these numbers are outside of the trigonometric 28


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function value range (-1 to +1) – the low water/corn oil interfacial tension is responsible. It is apparent, though, that the lower the right-hand side of Equation 2 is, the less wettable the solid surface is by liquid 1 under liquid 2, and therefore H+-CNC and NH(Et)3+-CNC films have the lowest wettability towards water under corn oil, followed by the Na+-CNC film, and K+-CNC film (i.e., the same trend in contact angle as for the hexadecane system). Experimentally, the H+ and NH(Et)3+-CNC three phase contact angles with corn oil are the same and higher compared to Na+ and K+-CNC films that are the same; in other words, the H+-CNCs and NH(Et)3+-CNCs are able to bend the oil-water interface more effectively. (Again we note that while TEA is added to neutralize the H+-CNC suspensions, the exact degree of ion exchange of H+ to NH(Et)3+ is unknown, and TEA-neutralized CNCs do not seem to be more hydrophobic; therefore the NH(Et)3+-CNCs may be more of a combination of H+-CNCs and NH(Et)3+-CNCs, hence their almost identical behavior to H+-CNCs.) These calculations and Figure 9c suggest that even small nuances in contact angle may change emulsion behavior significantly. Based on the Hofmeister series,51 these counterions (ignoring NH(Et)3+) are ordered K+>Na+>H+ in terms of their ability to increase water surface tension (and decrease solubility of non-polar species). It follows that ions high in the series should be less effective at stabilizing emulsions because a three phase contact angle close to 90o is sought after. The ability of H+-CNCs, Na+-CNCs and K+-CNCs follows this order (lower in the series stabilizes O/W emulsions better) indicating that the ability for ions to bind and structure water and promote hydrophobic interaction is at least partly responsible for the emulsion stability measured through these experiments.

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Figure 9. Contact angle of a 3 µL drop of water on a spin-coated CNC film (with H+, NH(Et)3+, Na+, and K+ counterions) as a function of time at 25℃ (a). Contact angle of a 3 µL drop of corn oil or hexadecane on a spin-coated CNC film (with H+, NH(Et)3+, Na+, and K+ counterions) as a function of time at 25℃ (b). Three-phase contact angle of a 3 µL drop of water on a spin-coated CNC film (with H+, NH(Et)3+, Na+, and K+ counterions) under corn oil as a function of time at 25℃ (c). Three-phase contact angle of a 3 µL drop of water on a spin-coated CNC film (with H+, NH(Et)3+, Na+, and K+ counterions) under hexadecane as a function of time at 25℃ (d). Inset images show the original shapes of liquid droplets on the surface of the CNC films at 0 min. Each point is an average of three measurements, and error bars represent the standard deviation. Lines drawn between points are provided to guide the eye.

CONCLUSION Overall, cellulose nanocrystals are amphiphilic and can stabilize emulsions despite not being highly surface active – their wettability and the ability of their counterion to interact with water and oils changes their emulsion behavior. The counterion has a significant effect, and while it is straightforward to exchange CNC counterions (reversibly) between H+, Na+ or K+, etc. the choice should not be overlooked when designing CNC-based products. In this work we demonstrated that acid-form CNCs (H+-CNCs) can stabilize corn oil-in-water Pickering emulsion without any additives or chemical modification. For lower viscosity and density oils, H+-CNCs do not perform as well, but the addition of 20 mM salt is 30


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sufficient to inhibit droplet coalescence, oil leakage and emulsion creaming. On their own, salt-form CNCs (Na+-CNCs and K+-CNCs) fail to stabilize emulsions over the long term. With the addition of salt, minimal change in emulsion droplet size occurs with storage, but droplet aggregation due to screened repulsion and density mismatch in the phases leads to extensive creaming. To avoid creaming, less salt and/or viscosity modifiers could be added. Interestingly, if the CNC suspension pH is changed with an organic weak base, or after the preparation of the emulsion, the same properties normally exhibited by H+-CNC-stabilized emulsions are retained. We believe this study highlights some of the challenges associated with preparing liquid formulated products such as emulsions, whereby the order in which components are added and how they are mixed affect the final properties. Similarly, even very small changes in wettability and interparticle/intermolecular interactions can drastically affect material performance and stability.

AUTHOR INFORMATION Corresponding Author *Emily D. Cranston. E-mail: [email protected] *Zhiping Mao. E-mail: [email protected] ACKNOWLEDGMENTS Funding support from the China Scholarship Council and NSERC Engage EGP507127‐16 with Dow are gratefully acknowledged.

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Effect of Counterion Choice on the Stability of Cellulose Nanocrystal

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