Langmuir 2009, 25, 497-502
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Phase Separation Behavior in Aqueous Suspensions of Bacterial Cellulose Nanocrystals Prepared by Sulfuric Acid Treatment Asako Hirai,* Osamu Inui, Fumitaka Horii, and Masaki Tsuji Institute for Chemical Research, Kyoto UniVersity, Uji, Kyoto 611-011, Japan ReceiVed September 8, 2008. ReVised Manuscript ReceiVed October 14, 2008 Phase separation phenomena of aqueous suspensions of cellulose nanocrystals have been studied for bacterial cellulose (BC) prepared by sulfuric acid hydrolysis. Suspensions at concentrations above 0.42 wt % separated into the isotropic and chiral nematic phases with a clear phase boundary. The shape and size distribution of BC nanocrystals in both the phases were determined by transmission electron microscopy (TEM) and atomic force microscopy (AFM). The surface charge density was determined by conductometric titration. The effects of added NaCl (0-5.0 mM) on the phase separation behavior of the aqueous suspensions were investigated for a fixed total cellulose concentration. The volume fraction of the chiral nematic phase had a minimum value at a NaCl concentration of ca. 1.0 mM. At NaCl concentrations ranging from 2.0 to 5.0 mM, the suspensions did not separate into two phases, but became entirely liquid crystalline. The size of the ordered domains in the anisotropic phase decreased with an increase in the NaCl concentration from 0 to 2.75 mM. At 2.75 mM, only tactoids were observed in the entire region. At 5.0 mM, chiral nematic domains were no longer observed. The chiral nematic pitch decreased with increasing concentration of added NaCl, reached a minimum value at approximately 0.75 mM, and then increased sharply with the NaCl concentration up to 2.0 mM.
Introduction It has been reported previously that an aqueous suspension of cellulose nanocrystals prepared by sulfuric acid treatment of cellulose microfibrils separates into two phases above a critical concentration.1 Sulfuric acid-hydrolyzed cellulose is often referred to as microcrystals, whiskers, nanocrystals, microcrystallites, or microfibrils. Hereafter, sulfuric acid-treated cellulose is called “cellulose nanocrystals”. The upper layer is isotropic, and the lower layer forms a chiral nematic liquid crystalline phase. The phase separation phenomenon and chiral nematic texture of cellulose nanocrystal suspensions for cotton and wood cellulose have been extensively studied.1-5 The properties and applications of cellulose nanocrystal suspensions were reviewed recently.6,7 Dong et al.3 reported the effects of the ionic strength on the isotropic-chiral nematic phase transition of suspensions of cotton cellulose nanocrystals; the volume fraction of the chiral nematic phase decreased from 0.56 to 0.05 as the concentration of the added electrolyte (HCl, NaCl, or KCl) increased from 0 to 2.4 mM. The added electrolytes induced a decrease in the chiral nematic pitch and an increase in the chiral twist power. Wood and cotton cellulose nanocrystals have been found to have a width of 3-5 nm and a length of 100-350 nm.5 Recently, Elazzouzi-Hafraoui et al.8 presented a more detailed analysis of the shape and size distribution of cellulose nanocrystals treated * To whom correspondence should be addressed. Phone: +81-774-38-3151. Fax: +81-774-38-3148. E-mail:
[email protected]. (1) Revol, J.-F.; Bradford, H.; Giasson, J.; Marchessault, R. H.; Gray, D. G. Int. J. Biol. Macromol. 1992, 14, 170–172. (2) Revol, J.-F.; Godbout, L.; Dong, X. M.; Gray, D. G.; Chanzy, H.; Maret, G. Liq. Cryst. 1994, 16, 127–134. (3) Dong, X. M.; Kimura, T.; Revol, J.-F.; Gray, D. G. Langmuir 1996, 12, 2076–2082. (4) Dong, X. M.; Revol, J.-F.; Gray, D. G. Cellulose 1998, 5, 19–32. (5) Beck-Candanedo, S.; Roman, M.; Gray, D. G. Biomacromolecules 2005, 6, 1048–1054. (6) De Souza Lima, M. M.; Borsali, R. Macromol. Rapid Commun. 2004, 25, 771–787. (7) Azizi Samir, M. A. S.; Alloin, F.; Dufresne, A. Biomacromolecules 2005, 6, 612–626. (8) Elazzouzi-Hafraoui, S.; Nishiyama, Y.; Putaux, J.-L.; Heux, L.; Dubreuil, F.; Rochas, C. Biomacromolecules 2008, 9, 57–65.
with 65% sulfuric acid for cotton, Avicel, and tunicate. With regard to bacterial cellulose (BC) nanocrystals, it was reported that the nanocrystals have rectangular cross sections or a ribbonlike shape with dimensions of ca. 10 nm × 50 nm and a length ranging from 100 nm to several micrometers, although these dimensions depend on the hydrolysis conditions.9 BC nanocrystals have significantly larger aspect ratios than wood and cotton nanocrystals. To the best of our knowledge, the phase separation behavior of BC nanocrystals has been reported in two papers. Revol et al.1 only mentioned that they observed the chiral ordering of an aqueous suspension for BC nanocrystals as well as wood, cotton, and ramie nanocrystals. Araki and Kuga9 reported that BC nanocrystals prepared by 65% sulfuric acid at 70 °C for 30 min underwent spontaneous nematic phase separation after complete desalination and that the addition of a trace electrolyte (0.1 mM NaCl) led to the formation of the chiral nematic phase. The presence of an electrolyte (0-1.0 mM) decreased the volume of the lower anisotropic phase significantly for a 1.58% cellulose suspension. A detailed examination of the phase separation behavior, however, was not carried out. In this study, the phase separation behavior of BC nanocrystals has been investigated in greater detail. The size of the nanocrystals in each layer is estimated by TEM and AFM. The surface charge density is determined by conductometric titration. The texture of the chiral nematic phase is compared with those of cotton and wood cellulose nanocrystals. The effects of added NaCl on the phase separation behavior of the aqueous suspension are investigated. The changes in the liquid crystalline textures upon the addition of salt are described and discussed.
Experimental Section Sample Preparation. Commercial food-grade BC was supplied by Fujicco Co. Ltd., Kobe, Japan. A large amount of BC, which was cut into the size of approximately 10 mm × 10 mm × 10 mm, was washed in running water for one week to remove the acetic acid added for preservation. To remove bacterial cell debris, BC was (9) Araki, J.; Kuga, S. Langmuir 2001, 17, 4493–4496.
10.1021/la802947m CCC: $40.75 2009 American Chemical Society Published on Web 12/04/2008
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Figure 1. Phase behavior of BC nanocrystals dispersed in deionized water as a function of the total concentration after 25 days of standing.
boiled in a 1 wt % NaOH aqueous solution for 2 h and then washed with water. This procedure was repeated, and BC was then neutralized with 0.2% acetic acid. Purified BC was homogenized using a multiblender mill (Nihonseiki Kaisha, Ltd., BLAS-501) and then mixed with sulfuric acid to a final sulfuric acid concentration of 60 wt % using an acid-to-cellulose ratio of 70 (mL/g); it was then stirred at 51 °C for 1 h. The sample was repeatedly washed with water by centrifugation, and the turbid supernatant was collected and thoroughly dialyzed against deionized water. The sample was passed through a mixed-bed ion-exchange resin column (Amberlite IRA 400JCL and IR120BHAG). The suspension thus obtained was concentrated by osmotic compression using cellulose dialysis tubing (Viskase Companies, Inc.) with molecular weight cutoffs of 12000-16000 and a 20% poly(ethylene glycol) (Mw ) 20000) solution. This was followed by sonication with 30 W (Branson Sonic Power Co., model 250) for 1 min and dilution with deionized water or a NaCl solution to obtain the desired cellulose concentrations (0.2-4.0 wt %) and NaCl concentrations (0-5 mM). Optical Measurements. The phase separation of the suspension was examined by viewing the sample glass vials between crossed polarizers. The volume fraction of each phase in the total suspension was obtained by measuring the height of each phase and the height of the total suspension in the vial. More specifically, the suspension sample was poured into a spectroscopic cell having a width, height, and path length of 10, 40, and 1 mm, respectively. Polarizing optical micrographs were obtained using a Nikon OPTIPHOTO2-POL polarizing microscope. Transmission Electron Microscopy (TEM). The size of the nanocrystals was determined using a JEOL JEM-200CS transmission electron microscope operated at 200 kV. A 3 µL sample of ca. 0.01 wt % suspension was placed onto a Formvar/carbon-coated Cu grid and negatively stained with 1% aqueous uranyl acetate containing Bacitracin having a concentration of 10-4 g/mL. Atomic Force Microscopy (AFM). AFM images were obtained at room temperature in the dynamic force mode (Seiko Instruments Inc., Chiba, Japan, SPA-400 SII controlled by SPI3800N) using an Olympus OMCL-AC160TS silicon cantilever (tip radius
