Biomacromolecules 2008, 9, 57–65
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The Shape and Size Distribution of Crystalline Nanoparticles Prepared by Acid Hydrolysis of Native Cellulose Samira Elazzouzi-Hafraoui,† Yoshiharu Nishiyama,*,† Jean-Luc Putaux,† Laurent Heux,† Frédéric Dubreuil,† and Cyrille Rochas‡ Centre de Recherches sur les Macromolécules Végétales (CERMAV-CNRS), BP 53, F-38041 Grenoble cedex 9, France - affiliated with Université Joseph Fourier and member of the Institut de Chimie Moléculaire de Grenoble, and Laboratoire de Spectrométrie Physique, UMR 5588, BP 87, F-38402 Saint Martin d’Hères, France Received July 11, 2007; Revised Manuscript Received September 25, 2007
The shape and size distribution of crystalline nanoparticles resulting from the sulfuric acid hydrolysis of cellulose from cotton, Avicel, and tunicate were investigated using transmission electron microscopy (TEM) and atomic force microscopy (AFM) as well as small- and wide-angle X-ray scattering (SAXS and WAXS). Images of negatively stained and cryo-TEM specimens showed that the majority of cellulose particles were flat objects constituted by elementary crystallites whose lateral adhesion was resistant against hydrolysis and sonication treatments. Moreover, tunicin whiskers were described as twisted ribbons with an estimated pitch of 2.4–3.2 µm. Length and width distributions of all samples were generally well described by log-normal functions, with the exception of tunicin, which had less lateral aggregation. AFM observation confirmed that the thickness of the nanocrystals was almost constant for a given origin and corresponded to the crystallite size measured from peak broadening in WAXS spectra. Experimental SAXS profiles were numerically simulated, combining the dimensions and size distribution functions determined by the various techniques.
Introduction It has been known for more than half a century that stable nanoparticle suspensions could be prepared by submitting native cellulose to a harsh sulfuric acid hydrolysis often followed by ultrasound treatments.1 Such nanoparticles, prepared from several types of higher plant cellulose with different hydrolysis conditions, have been characterized by using methods such as transmission electron microscopy, sedimentation, flow birefringence, and viscometry.1,2 It was generally concluded that the particles were elongated and flat, a few hundreds of nanometers long, 10–20 nm wide, and a few nm thick.2,3 Nanoparticles from acid-hydrolyzed cellulose and chitin attracted a renewed interest when it was shown that, in suspension, they could form chiral nematic phases4,5 resembling the highly textured organizations of microfibrils found in native systems.6,7 The effects of hydrolysis conditions and surface charge density,8,9 ionic strength,10–14 and pH10,15 on the phase separation behavior and structures of the colloidal system have been extensively studied over the last 15 years. However, despite the previous observation that the particles were rather flat objects, they have usually been treated as cylinders with mean diameters and lengths in models used to describe their phase separation behavior in suspension.9 In fact, for aqueous suspensions stabilized by electrostatic repulsion at low ionic strength, the exact shape of the particles mattered little because the effective diameter generated by the repulsive charges on the surface was substantially larger than the particle width.9 Steric stabilization of the nanocrystals with either surfactants16 or surface chemical modification17 recently allowed studying cellulose whiskers suspensions in apolar solvents and media with * Corresponding author. E-mail:
[email protected]. Fax: +33 476547203. † Centre de Recherches sur les Macromolécules Végétales. ‡ Laboratoire de Spectrométrie Physique.
high salinity. As higher concentrations could be achieved, the shape and size of the particles would play a more significant role on the self-organization properties. Nanoparticles with different morphologies can be prepared by varying the source of cellulose and the hydrolysis conditions. For instance, whiskerlike particles with a length of the order of micrometers are obtained by hydrolyzing highly crystalline cellulose samples from tunicates18 and green algae,19 whereas higher plant cellulose from cotton and wood pulp yields shorter particles a few hundreds of nanometers long. For a given system, the dimensions of the cellulose nanoparticles were generally measured using imaging and/or scattering techniques.20 In some cases, size distribution histograms were determined from transmission electron micrographs,2,7 but the nature of the distribution has rarely been studied in detail. To our knowledge, the size distribution was only taken into account in one case, reported by Marchessault et al., who simulated the birefringence properties of Ramie crystallites.2 In the study presented here, we submitted three sources of cellulose to sulfuric acid hydrolysis. The shape and size distribution of the resulting nanoparticles were determined from transmission electron microscopy (TEM) images, and height measurements were performed using atomic force microscopy (AFM). Small- and wide-angle X-ray scattering (SAXS and WAXS) experiments were used to characterize the whole colloidal system.21
Experimental Section Cellulose Sources. Three sources of cellulose were used. Cotton linters were provided by Rhône-Poulenc Tubize Plastics (Belgium) and used without any further purification. Avicel, a commercial microcrystalline cellulose resulting from the hydrochloric acid hydrolysis of wood pulp and containing 20 µm particles, was purchased from FMC Europe NV, Belgium. Tunicin, the cellulosic mantle of Halocynthia
10.1021/bm700769p CCC: $40.75 2008 American Chemical Society Published on Web 12/04/2007
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Biomacromolecules, Vol. 9, No. 1, 2008
Elazzouzi-Hafraoui et al.
Figure 1. TEM micrographs of negatively stained cellulose whiskers obtained by sulfuric acid hydrolysis of cotton (a), Avicel (b), and tunicate (c-e) cellulose. Insets: enlarged views of some characteristic particles. The arrows in (d) indicate zones where the whiskers are seen edge-on.
roretzi, a sea animal, was purified by alternating treatments with KOH and NaClO2.22 Preparation of Cellulose Whiskers Suspensions. Cotton linters were hydrolyzed according to the method described by Revol et al.4 They were treated with 65% sulfuric acid during 30 min at four different temperatures, namely 45, 54, 63, and 72 °C. In the following, the resulting samples will be referred to as Cot45, Cot54, Cot63, and Cot72, respectively. The suspensions were washed by centrifugation, dialyzed to neutrality against distilled water, and ultrasonicated for 4 min with a rod-type sonicator (Branson Sonifier B12). This treatment heated up the suspension by about 20 °C. After these treatments, the suspensions were filtered on 8 µm then 1 µm cellulose nitrate membranes (Sartorius) and stocked with mixed bed resin (Sigma TMD-8) in order to eliminate residual electrolytes. Avicel was hydrolyzed at 72 °C following the same procedure. The resulting sample will be referred to as Avi72. Tunicin was treated with 48% sulfuric acid during 13 h at 55 °C. Acidfree microcrystalline cellulose suspensions were obtained after repeated centrifugations, dialysis to neutrality against distilled water, and ultrasonication for 1 min. This suspension, referred to as Tun55 in the following, was stocked with mixed bed resin. Transmission Electron Microscopy. Drops of 0.001 wt % cellulose microcrystal suspensions were deposited on glow-discharged carboncoated TEM grids. The specimens were then negatively stained with 2% uranyl acetate, prior to complete drying, and observed using a Philips CM200 electron microscope operating at 80 kV. Images were recorded on Kodak SO163 films. Selected negatives were digitized using a Kodak Megaplus CCD camera. For each preparation, the width and length of about 1000 particles (except tunicin, for which 200 particles were counted) were measured from the TEM images by using the AnalySIS software. Thin vitrified films of 0.1 wt % cellulose microcrystal suspensions were also prepared by quench-freezing in liquid ethane by using a procedure described elsewhere.23 They were observed by cryo-TEM, at low temperature (-180 °C), in a Gatan 626 cryoholder. Atomic Force Microscopy. After a short sonication to prevent the formation of aggregates, drops of dilute cellulose microcrystals suspensions were deposited onto freshly cleaved mica. After 30 min, the excess liquid was removed and the remaining film allowed to dry. AFM observations were carried out using a Molecular Imaging Pico
Plus microscope operating in air and intermittent contact mode with a Micromash NC36 tip. Wide-Angle X-ray Scattering. Concentrated cellulose suspensions (3–4 wt %) were allowed to dry onto flat Teflon surfaces. At this concentration, the suspension of Avicel particles was more viscous than those containing cotton or tunicin microcrystals. The resulting films were X-rayed with a Ni-filtered Cu KR radiation (λ ) 1.542 Å), using a Philips PW3830 generator operating at 30 kV and 20 mA. The films were positioned either parallel or perpendicular to the X-ray beam. Diffraction patterns were recorded on Fujifilm imaging plates, read with a Fujifilm BAS-1800II bioimaging analyzer. Diffraction profiles were obtained by radially integrating the intensity over fan-shaped portions of the spectra. The diffraction peaks were fitted with pseudo-voigt peak functions, assuming a linear background. The dimension of the crystal perpendicular to the diffracting planes with hkl Miller indices, Dhkl, was evaluated by using Scherrer’s expression:
Dhkl )
0.9 × λ β1/2 × cos θ
(1)
where θ is the diffraction angle, λ the X-ray wavelength and β1/2 the peak width at half of maximum intensity.24 Small-Angle X-ray Scattering. SAXS experiments were performed on the BM02 beamline of the European Synchrotron Radiation Facility (Grenoble, France). Dilute cellulose suspensions (
