Article pubs.acs.org/Biomac
Polymer-Grafted Cellulose Nanocrystals as pH-Responsive Reversible Flocculants Kevin H. M. Kan, Jian Li, Kushlani Wijesekera, and Emily D. Cranston* Department of Chemical Engineering, McMaster University, Hamilton L8S 4L7, Canada S Supporting Information *
ABSTRACT: Cellulose nanocrystals (CNCs) are a sustainable nanomaterial with applications spanning composites, coatings, gels, and foams. Surface modification routes to optimize CNC interfacial compatibility and functionality are required to exploit the full potential of this material in the design of new products. In this work, CNCs have been rendered pH-responsive by surface-initiated graft polymerization of 4-vinylpyridine with the initiator ceric(IV) ammonium nitrate. The polymerization is a one-pot, water-based synthesis carried out under sonication, which ensures even dispersion of the cellulose nanocrystals during the reaction. The resultant suspensions of poly(4-vinylpyridine)-grafted cellulose nanocrystals (P4VP-g-CNCs) show reversible flocculation and sedimentation with changes in pH; the loss of colloidal stability is visible by eye even at concentrations as low as 0.004 wt %. The presence of grafted polymer and the ability to tune the hydrophilic/hydrophobic properties of P4VP-g-CNCs were characterized by Fourier transform infrared spectroscopy, elemental analysis, electrophoretic mobility, mass spectrometry, transmittance spectroscopy, contact-angle measurements, thermal analysis, and various microscopies. Atomic force microscopy showed no observable changes in the CNC dimensions or degree of aggregation after polymer grafting, and a liquid crystalline nematic phase of the modified CNCs was detected by polarized light microscopy. Controlled stability and wettability of P4VP-gCNCs is advantageous both in composite design, where cellulose nanocrystals generally have limited dispersibility in nonpolar matrices, and as biodegradable flocculants. The responsive nature of these novel nanoparticles may offer new applications for CNCs in biomedical devices, as clarifying agents, and in industrial separation processes.
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tion,13 cationization,14 silylation,15 polymer grafting,16−18 and so on.19,20 Steric stabilization with surface-grafted polymer brushes has been particularly effective in improving the stability and dispersibility of CNCs in nonpolar solvents and polymer matrices.21,22 Unfortunately, these reactions are generally performed in organic media where tedious and lengthy solvent exchange processes are required and CNCs are, at best, partially agglomerated.1,20 We present an approach to functionalize CNCs in water, where they are colloidally stable and welldispersed, because we believe that nanoparticles must remain individualized during modification reactions to achieve a reproducible and uniform material as the product. The grafting of polymers onto the surface of CNCs can be achieved through both “grafting-to” and “grafting-from” techniques. 23 “Grafting-to” involves the attachment of presynthesized (and characterized) polymer chains to the surface hydroxyl or sulfate ester groups. Alternatively, “graftingfrom” involves the surface initiated in situ polymerization from initiators, which are normally immobilized on the CNC surface, to generate tethered polymers by conventional free radical,24 ionic,25 or ring-opening polymerization reactions.26,27 In contrast, we are reporting a system where polymer chains are
INTRODUCTION In recent years, cellulose nanocrystals (CNCs) have attracted significant attention not only because of their renewable source and biodegradability but also because of their low density, high aspect ratio, high tensile strength, and unique optical properties.1−5 CNCs are now being produced in industrially relevant quantities in both Canada and the USA.6 These whisker-shaped nanoparticles can be produced from a variety of natural cellulose sources and have dimensions of a few nanometers wide by hundreds of nanometers long.1 CNCs are generally isolated by acid hydrolysis which removes the amorphous regions of cellulose and leaves behind the highly crystalline regions that are less accessible to acid degradation.7−9 Because the crystalline cellulose is not dissolved during the hydrolysis, CNCs are composed of cellulose in the native cellulose I crystal form and have a degree of crystallinity of ∼90%; the other 10% is attributed to defects and disordered surface polymer chains.10 The controlled hydrolysis of cellulose with concentrated sulfuric acid yields an aqueous suspension of particles that are colloidally stabilized via double-layer repulsive forces due to surface-grafted sulfate half ester groups that are anionic under most solution conditions.9,11,12 CNCs can be modified along their crystalline backbone through hydroxyl substitution reactions to change the surface chemistry. Previously reported surface modifications include esterification, sulfonation, oxida© 2013 American Chemical Society
Received: May 25, 2013 Revised: July 12, 2013 Published: July 18, 2013 3130
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up a range of biomedical and industrial separation opportunities for pH-responsive CNC-based materials.
grafted from CNC surface hydroxyl groups without the need for a prepolymerization step to attach initiators to the surface. We use the ceric−cerous redox system to initiate polymerization from the backbone of cellulose. Free radicals are formed when single electrons are transferred from cellulose hydroxyl groups to the ceric initiator.28,29 Mino and Kaizerman30 were the first to discuss graft polymerization using ceric ions, while Schwab et al.31 extended this to cellulose. Since then, the grafting of various vinyl monomers using this technique has been applied to numerous cellulosic materials,28,32−35 including cellulose nanofibrils.36 However, like most chemistries that target cellulose, the grafting reaction with ceric ions is believed to mainly occur in amorphous cellulose regions due to higher accesibility.33 For CNCs with a surface area of several hundred square meters per gram,37 there is a huge number of accessible surface cellulose chains, implying that ceric-ion grafting may be effective on the surface of the nanoparticles but that radical formation inside the CNCs is unlikely. This is the first research article describing the use of ceric-ion-initiated polymerization with purely crystalline cellulose and moreover applied to CNCs. This work coincides with a recent patent by Hamad and Su that also describes “grafting-from” CNCs using ceric ammonium nitrate.38 The responsiveness of polymers to triggers such as pH,39,40 temperature,22,34,41,42 and light43 is relevant for biotechnological applications and “smart” material design, such as drug delivery and biosensors as well as construction materials, textiles, coatings, miniaturized devices, and industrial processing.44 Poly(4-vinylpyridine) (P4VP) is one such responsive polymer that undergoes chemical and morphological changes with pH as the cue. P4VP is hydrophilic in its protonated state (pH 5) P4VP becomes hydrophobic and precipitates from aqueous solution.45,46 Previous reports of stimuli-responsive polymers attached to CNCs include thermosensitive amine-terminated statistical polymers (Jeffamines),47 poly(N-isopropylacrylamide) (PNIPAM),22,48,49 and poly(ethylene glycol) (PEG).42 The grafting of single-stranded oligonucleotides to CNCs has also resulted in responsive, self-assembling nanostructures that can be disassembled by increasing the temperature above the melting point of duplexed DNA.50 To our knowledge, the current research is the first report of using a pH-responsive polymer grafted from CNCs. Furthermore, the response with P4VP is a reversible flocculation and sedimentation of CNCs from suspension, which is visible by eye at extremely low concentrations. Our intent in this work has been to shift toward more industrially viable surface modification routes for CNCs by using a one-pot, water-based synthesis to produce a low-cost, green, and functional material. Sonication is used throughout the polymerization reaction to ensure that individual CNCs are surface-functionalized and aggregation is avoided; we find that reproducible and uniform material properties are achieved as a result. The ceric-ion-initiated radical polymerization gives P4VP-grafted cellulose nanocrystals (P4VP-g-CNCs) that change character from cationic to anionic and hydrophilic to hydrophobic as the pH of the suspension is increased. Importantly, this change is reversible, and flocculated systems return to being colloidally stable when the pH is decreased. These controlled properties offer new potential to achieve a uniform dispersion of CNCs in hydrophobic matrices and open
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EXPERIMENTAL SECTION
Materials. 4-Vinylpyridine (4VP), cerium(IV) ammonium nitrate (CAN), inhibitor removers, and P4VP (Mw ≈ 60 000 g/mol) were purchased from Sigma-Aldrich. Nitric acid (70%) was obtained from EMD Chemicals and polyallylamine hydrochloride (PAH, Mw = 120 000−200 000 g/mol) was purchased from Polysciences. Preparation of Cellulose Nanocrystals. Suspensions of CNCs were prepared by sulfuric acid hydrolysis, as previously described.51 Typically, 40 g of cotton filter aid (Whatman ashless filter aid, GE Healthcare Canada, Mississauga, ON, Canada) was treated with 700 mL of 64 wt % sulfuric acid (Fischer Scientific) at 45 °C for 45 min with constant stirring. Following the acid hydrolysis, the suspension was diluted 10-fold with purified Type I water with a resistivity of 18.2 MΩ × cm (Barnstead NANOpure DIamond system, Thermo Scientific, Asheville, NC) to quench the reaction. The suspension was then centrifuged repeatedly at 6000 rpm for 10 min to concentrate the cellulose, while excess water and acid were removed. The resulting precipitant was rinsed in purified water, recentrifuged, and dialyzed in purified water until constant, neutral pH was achieved in the dialysis reservoir. The suspension was sonicated three times for 15 min intervals (Sonifier 450, Branson Ultrasonics, Danbury, CT) at 60% output while cooling in an ice bath to prevent overheating. Dowex Marathon C hydrogen form resin (Sigma-Aldrich) was added to the cellulose suspension for 3 days and removed by filtering through glass microfiber filters (Whatman grade GF/B, VWR, Chicago, IL). The resulting suspension was ∼1% cellulose by weight. The surface charge density of the CNCs was calculated to be 0.34 ± 0.01 e/nm2 from elemental analysis, with average crystal dimensions of 122 × 8 nm from atomic force microscopy (AFM) height images. Graft Polymerization. Suspensions of P4VP-g-CNCs were prepared as follows. First, the 4VP monomer was passed through an inhibitor removal column. Next, 40 mL of a 1 wt % CNC suspension, 0.75 mL of 70 wt % HNO3 (EMD Chemicals), and 1.17 mL of 4VP were added to a 50 mL round-bottomed flask. Under a N2 atmosphere, the mixture was sonicated continuously at 60% output for 45 min in an ice bath, at which point 131 mg of CAN was dissolved in 1 mL of purified water and added to the suspension. (An inert N2 atmosphere was used to avoid oxygen induced inactivation of the radicals, as suggested by McDowall et al.28) The mixture was sonicated for another 2 h in an ice bath. The mixture was then left stirring overnight under a N2 atmosphere. The following day, the mixture was ultracentrifuged at 50 000 rpm for 1 h and washed with acidic purified water (pH 2) until the UV absorbance of the supernatant at ∼252 nm had decreased to 0.2 to 0.7 absorbance units, implying that monomer and homopolymer had been removed from the suspension. (4VP strongly absorbs in the UV region.) Sonication was applied between each centrifugation step to redisperse the nanoparticles and ensure efficient removal of unreacted reagents. The P4VP-g-CNC suspension was filtered through glass microfiber filter paper to remove any metal particles from the sonication probe and extensively dialyzed against acidic water (pH 2). After dialysis, the P4VP-g-CNC suspension was ultrafiltrated at 3500 rpm with 100 kDa ultrafiltration tubes for 15 min to yield a 1 wt % P4VP-g-CNC suspension, which was then stored in the refrigerator. Concentrated solutions of HCl (LabChem) and NaOH (Fischer Scientific) were used to adjust the pH of the suspensions to test the responsive behavior. Fourier-Transform Infrared (FTIR) Spectroscopy. Suspensions of CNCs and P4VP-g-CNCs were freeze-dried and incorporated into KBr pellets at ∼1 wt %, as was done for a neat P4VP sample. Furthermore, KBr pellets were dried in the oven at 150 °C for 10 min. FTIR spectra were recorded in transmission mode on a Nicolet 6700 FTIR spectrometer (Thermo Scientific). Elemental Analysis. Carbon, hydrogen, nitrogen, and sulfur content by mass for freeze-dried CNCs and P4VP-g-CNCs were determined by Micro Analysis (Wilmington, DE). The samples were combusted in a pure oxygen environment where the product gases 3131
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were separated and detected by thermal conductivity. Duplicates were measured for each sample, and averages are reported. Atomic Force Microscopy. Polished silicon wafers (Grinm Semiconductor Materials, Beijing, China) were cleaned in ethanol and cut into 1 cm × 1 cm squares and dipped into a 0.1 wt % PAH solution for 1 h and rinsed in purified water to adsorb a cationic precursor layer on the substrate. The silicon wafers were then dipped in either 0.001 wt % CNC or P4VP-g-CNC suspensions for 1 h and dried with compressed air. The nanoparticle-coated silicon wafers were imaged by AFM using a Nanoscope IIIa multimode scanning probe microscope with an E scanner (Bruker AXS, Santa Barbara, CA). Tapping mode images were collected in air with silicon cantilevers (AC 160TS, Olympus Canada, Richmond Hill, ON, Canada). The length and height of the CNCs were determined from cross-sectional analyses of AFM height images. The reported error intervals are confidence intervals calculated from the standard deviation (Sx) of repeat measurements (N), that is, Δx = Sx × t-value/(N)1/2, where the t-value is the Student’s t-distribution at a confidence level of 95% for N − 1 degrees of freedom. Over 100 individual CNCs were measured. Dynamic Light Scattering. Dynamic light scattering (DLS) experiments were carried out with a Malvern Zetasizer Nano-S instrument (Malvern, Worcestershire, U.K.) with a detection angle of 173°. Suspensions of CNCs and P4VP-g-CNCs at pH 4.5 and 2.5, respectively, were filtered through a Millipore Millex-FH nylon syringe filter (0.45 μm pore size). Measurements were obtained at 0.05 wt % concentrations, with 5 mM NaCl (Sigma-Aldrich) added, at 25 °C. The reported error intervals represent the standard error of the average of 3 measurements with 15 cycles per measurement. Electrophoretic Mobility. The electrophoretic mobility was measured using a Zeta Potential ZetaPlus analyzer (Brookhaven, Holtsville, NY). All samples were measured at 0.25 wt % concentrations, with 5 mM NaCl added, at 25 °C. All electrophoretic mobility data points include error bars, which represent the confidence intervals for an average of 10 measurements with 15 cycles per measurement. (In some cases the error bars are smaller than the symbols and are not necessarily visible.) Transmittance. The transmittance of all suspensions was measured using a DU800 UV−vis spectrometer (Beckman Coulter) at a wavelength of 500 nm. The concentration of the CNC and P4VPg-CNC suspensions were 0.25 wt % with 5 mM NaCl added. P4VP solutions for turbidity measurements were 0.25 wt % in 99.7% acetic acid (Caledon Laboratories Ltd.). Contact Angle. Static water contact-angle measurements were conducted on an NRL contact-angle goniometer, model 100-00-115 (Ramé-hart, Succasunna, NJ) equipped with a Sanyo VC8−3512T camera. Uniform films for contact-angle measurements were obtained by spin coating (Chemat Technology KW-4A, Northridge, CA) ∼10 wt % CNC and P4VP-g-CNC suspensions onto PAH-coated Si wafers at 4000 rpm for 60 s, followed by heat treatment for 12 h at 80 °C. Post-spin-coating, films were immersed in adjusted pH solutions of pH 2, 5, and 10 for 1 h and then dried at 50 °C for 1 h. Contact angles were measured with 5 μL droplets of purified water at pH 2, 5, and 10 on the films that had been preconditioned at the same pH value. Thermogravimetric Analysis. Thermogravimetric analysis (TGA) measurements were performed using a thermoanalyzer (Netzsch STA-409, Burlington, MA). 20−25 mg of freeze-dried P4VP, CNCs and P4VP-g-CNCs was heated to 500 °C under air with a 5 °C/min heating rate. Mass Spectrometry. Two types of mass spectrometry (MS) were used to analyze P4VP grafted from CNCs: (1) laser desorption/ ionization time-of-flight mass spectrometry (LDI-TOF MS) was conducted on the Micromass MALDI MicroMX spectrometer (Waters, Milford, MA) in linear mode from dried suspensions without an ionizing matrix and (2) direct-sample analysis time-of-flight mass spectrometry (DSA-TOF MS) was conducted on the AxION DSA System (Perklin Elmer, Waltham, MA) by applying 10 μL of each sample to the DSA rail mesh holder. Optical Microscopy. Photomicrographs of P4VP-g-CNC suspensions were taken with a polarized light microscope (Olympus BX51) equipped with a QImaging Retiga-EXi camera. A few drops of 1.0 wt %
P4VP-g-CNC suspension were placed on a standard glass microscope slide and covered with a coverslip. Pictures were taken as the P4VP-gCNC suspension evaporated from the edge of the slide.
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RESULTS AND DISCUSSION Responsive Poly(4-vinylpyridine)-Grafted Cellulose Nanocrystals. P4VP-g-CNCs were prepared using a surfaceinitiated “grafting-from” reaction with CAN. This one-pot, water-based synthetic route is illustrated in Scheme 1a. While Scheme 1. (a) Graft Polymerization of Poly(4-vinylpyridine) with Ceric(IV) Ammonium Nitrate Initiator from the Surface of Sulfuric Acid-Hydrolyzed Cellulose Nanocrystals and (b) the Generally Accepted Mechanism for the Formation of a Radical Site on Cellulose through Ceric Ion Reduction (Adapted from ref 32)
the exact mechanism is still debated,28,29 research suggests that the ceric ion forms a chelated complex with the cellulose molecule in which a single electron is transferred from cellulose to Ce(IV), breaking a cellulose C−C bond and forming a radical site where graft polymerization is initiated.34 Several possible sites exist where this oxidation can occur28,52 and the rate of oxidizing hemiacetal groups at the end of the cellulose chains is known to be the fastest;53 however, because of the low abundance of chain ends, the C2−C3 position is generally accepted as the major oxidation site,54 as shown in Scheme 1b.32 Extensive purification was performed prior to all analyses to remove any unbound homopolymer and to ensure that the detected polymer moieties were attached to the CNCs. The final pH of the P4VP-g-CNC suspension was 2.5 and showed no visible sedimentation, suggesting that a stable aqueous suspension was obtained under acidic conditions. The presence of P4VP in the P4VP-g-CNC sample was evidenced by FTIR spectroscopy by comparing to the spectra of pure P4VP and pure CNCs, as shown in the Supporting Information (Figure S1). Because the link between cellulose and P4VP was not unique when compared with the starting materials, we could not confirm that P4VP was covalently bound to CNCs. We merely conclude that P4VP was still existent after dialysis and ultrafiltration and further characterization was undertaken to 3132
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better understand the nature of the interaction between the nanoparticle and the polymer. P4VP is a weak polyelectrolyte with a pKa of ∼5.55,56 At pH >5, P4VP becomes hydrophobic and precipitates from aqueous solution due to the deprotonation of the pyridyl group.45,46 Suspensions of P4VP-g-CNCs also flocculate and sediment above pH 5, as shown macroscopically in Figure 1. Initially,
Figure 2. Effect of pH on the electrophoretic mobility of unmodified CNCs (◇) and P4VP-g-CNCs (■) in aqueous suspension.
nated pyridyl groups. At pH values above 5, the pyridyl group is deprotonated and the polymer chains collapse. When P4VP is in its collapsed uncharged state with stacked aromatic groups, P4VP-g-CNCs display a negative electrophoretic mobility approaching that of unmodified CNCs. For P4VP-g-CNCs at high pH, the electrophoretic mobility values alone imply that an electrostatically stable colloidal suspension should be present. The flocculation observed in Figure 1 must therefore be a result of the P4VP-g-CNCs becoming more hydrophobic and incompatible with the aqueous environment. Additionally, the negative surface charge above pH 5 is attributed to the CNCs’ sulfate ester groups, which are still present after the grafting reaction (see Grafting Density and Elemental Analysis section), which suggests that the polymer grafting density and the length of the P4VP polymer chains is not sufficient to completely cover, or screen, the native surface chemistry of the underlying CNCs. Light transmittance at 500 nm for a P4VP solution and suspensions of unmodified and polymer-grafted CNCs was also used to characterize the material’s response to pH (Figure 3). At pH values less than 4, the transmittance for the P4VP solution is high. At pH 5, the transmittance sharply decreases due to precipitation of the hydrophobic P4VP. As shown in the inset of Figure 3, this precipitation transforms the P4VP solution from transparent to opaque due to the formation of polymer particles that scatter visible light, but the polymer does not sediment over the time period of the measurement. This decline in transmittance is mirrored in the P4VP-g-CNC suspensions; however, the decrease is less sharp and the transition occurs over a pH range of 3−6. Conversely, the transmittance of the unmodified CNC suspension does not change over the whole pH range. The transmittance of P4VP-g-CNC suspensions as a function of time is presented in the Supporting Information (Figure S2a) and shows that the transmittance decreases for P4VP-gCNC suspensions above pH 5 over the first 300 min as the nanoparticles flocculate but then stabilize. At pH values less than 5, the transmittance is stable as a function of time, indicating that the hydrophilic nature and repulsive interactions between cationic pyridyl groups are able to stabilize P4VP-gCNC suspensions. The reversibility of this transition can also be followed by transmission measurements (Supporting Information, Figure S2b); however, agitation, mixing and
Figure 1. Photographs of CNC suspensions (0.25 wt %) (a) immediately after pH adjustment and (b) 1 h after pH adjustment; P4VP-g-CNC suspensions (0.25 wt %) (c) immediately after pH adjustment and (d) 1 h after pH adjustment. P4VP-g-CNCs flocculate and sediment above the pKa of P4VP.
both the unmodified and polymer-grafted CNCs appear stable as a function of pH (Figure 1a,c). After 1 h, however, the unmodified CNC suspensions remain insensitive to pH (Figure 1b), whereas the P4VP-g-CNCs above pH 5 are settled at the bottom of the vials (Figure 1d). The onset of flocculation is visible within a few minutes but continues to sediment over many hours. This transition is reversible; shifting the pH to values
