Article pubs.acs.org/IECR
Comparing Polymer-Supported TEMPO Mediators for Cellulose Oxidation and Subsequent Polyvinylamine Grafting Shuxian Shi,† Robert Pelton,*,‡ Qiang Fu,‡ and Songtao Yang‡ †
Key Laboratory of Carbon Fiber and Functional Polymers (Ministry of Education), Beijing University of Chemical Technology, Beijing 100029, China ‡ Department of Chemical Engineering, McMaster University, Hamilton, Ontario L8S 4L8, Canada ABSTRACT: A new poly(acrylic acid)-grafted TEMPO (PAA-T) is compared to polyvinylamine-grafted TEMPO and free TEMPO as oxidation mediators for cellulose. The polymer-immobilized mediators require lower overall TEMPO concentrations and they restrict oxidation to the exterior surfaces of porous cellulose. On the other hand, the resulting surfaces are coated with grafted polyvinylamine and laccase, if the enzyme is used as the primary oxidant. PAA-T is anionic and does not sufficiently adsorb onto anionic cellulose to give oxidation, whereas cellulose rendered cationic by an adsorbed layer of PVAm is oxidized by PAA-T + laccase. There is no clear “best choice” mediator/primary oxidant combination for cellulose oxidation subsequent to PVAm grafting; the advantages of each mediator are summarized.
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INTRODUCTION As the most abundant organic material on the planet, cellulose must be a cornerstone of any strategy for the transformation to a bio-based economy. From a materials perspective, this means expanding the property landscape of cellulosic materials. However, cellulose surfaces lack reactive functional groups and are hydrophilic, making them difficult to react and to adhere to, particularly when wet. To this end, we have been exploring new approaches to surface modification of cellulose fibers by covalently bonding polymeric primer layers onto cellulosic surfaces, making them more adhesive and reactive. Specifically, we have focused on the covalent attachment of polyvinylamine (PVAm) to cellulose by slightly oxidizing cellulose to give surface aldehyde groups that form imine and aminal linkages with polyvinylamine under mild conditions.1,2 PVAm was chosen to form the primer layer because PVAm has the highest content of primary amines of any commercial polymer. The resulting cellulose surfaces have grafted PVAm coverages in the range 0.1−1 mg/m2. Because of the high PVAm amine content, a 1 mg/m2 grafted layer corresponds to a primary amine density of ∼0.1 nm2 per amine. Such highly functionalized cellulose surfaces are cationic, adhesive, and chemically reactive. The mild oxidation of cellulose to produce surface aldehyde groups is a critical part of our approach. Initially, we employed Isogai’s cellulose oxidation recipe based on NaClO/NaBr in the presence of a TEMPO mediator.3 The generally accepted mechanism is that the NaClO oxidizes TEMPO to the corresponding oxoammonium ion that shuttles to a primary alcohol to form a covalent transition state. This sequentially oxidizes the alcohol to the corresponding aldehyde and then to the carboxylic acid.4−10 The TEMPO + NaClO/NaBr oxidation has two practical limitations. First, the reaction must be conducted at pH 10−11, which is not compatible with papermaking technologies. Second, TEMPO is expensive and with toxicological11 impacts; therefore, processes involving large volumes of aqueous © 2014 American Chemical Society
TEMPO solutions offer engineering challenges. Instead, we have focused on variations of TEMPO mediated oxidations where the TEMPO mediator is grafted onto water-soluble polymers. Initially, we grafted TEMPO onto PVAm, giving a polymer, PVAm-T (see structures in Figure 1) that filled two roles.12,13 First, cationic PVAm-T spontaneously adsorbs onto cellulose, concentrating the TEMPO moieties near the cellulose surface. Second, the PVAm-T condenses with aldehydes giving the desired covalently bonded primer layer. Herein we report a new variation of immobilized TEMPO, PAA-T, where TEMPO is grafted to poly(acrylic acid), giving an anionic TEMPO copolymer (see structure in Figure 1). This new mediator differs from PVAm-T in that PAA-T is an anionic polyelectrolyte, whereas PVAm-T is highly cationic. In addition, there are no functional groups on PAA-T that will react with aldehyde groups generated on the cellulose. The objective of this paper is to report a comparative study of three cellulose oxidation mediators: TEMPO, polyvinylamine-graft-TEMPO (PVAm-T), and poly(acrylic acid)-graft-TEMPO (PAA-T) with respect to grafting PVAm onto cellulose. To facilitate this comparison, Figure 1 summarizes the four combinations of mediators and primary oxidants discussed below.
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EXPERIMENTAL SECTION
Materials. Polyvinylamine (PVAm), with a molecular weight of 45 kDa and a hydrolysis degree of 75%, was obtained from BASF, Ludwigshafen (Lupamin 5095) and was purified by dialysis in water and freeze-dried before use. Poly(acrylic acid) solution with a weight-average molecular weight of about 100 000 and concentration of 35 wt % in water was purchased from Sigma-Aldrich. It was used without further purification.
Received: Revised: Accepted: Published: 4748
January 20, 2014 February 22, 2014 March 3, 2014 March 3, 2014 dx.doi.org/10.1021/ie500280e | Ind. Eng. Chem. Res. 2014, 53, 4748−4754
Industrial & Engineering Chemistry Research
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In a typical experiment, 2.0 g of poly(acrylic acid) solution (35 wt %) and 2.0 g of EDC were dissolved in 200 mL of deionized water in a 500 mL beaker fitted with a pH electrode. After 30 min, 0.40 g of 4-amino-TEMPO was added into the solution. The pH of the solution was continuously maintained at 4.75 for 4 h by manual addition of 0.1 M HCl or 0.1 M NaOH. Afterward, the reaction was stirred overnight. The resulting solution was dialyzed (Spectra/Pro 1, MWCO 6000− 8000) against deionized water for 2 weeks. The final product was freeze-dried and stored in a desiccator. Conductometric Titration. The carboxyl contents for PAA-T were determined by simultaneous potentiometric and conductometric titration. Thirty milligrams of the freeze-dried sample was dissolved in 80 mL of 5 mM NaCl. The initial pH was adjusted to 3 with 1.0 M HCl and then was titrated with 0.1 M NaOH at 25 °C. The grafted TEMPO contents were calculated from the change in carboxyl contents with TEMPO grafting. Polyelectrolyte Titrations. The net charge contents of PAA-TEMPO and laccase were measured by polyelectrolyte titration (Mütek PCD titrator) in 50 mM sodium acetate buffer at a pH of 5. A 1 mM solution of poly(diallyldimethyl ammonium chloride) was used to titrate PAA-T (0.1 g/L) and laccase (0.1 g/L) and the end point was taken as the titrant volume corresponding to zero streaming current.14 We note that under these relatively high electrolyte concentrations, polyelectrolyte titration end points are not necessarily stoichiometric.15 Laccase Activity Assay. The activity of laccase solutions was measured by an assay based on the oxidation of ABTS.16 Laccase was dissolved in a 50 mM sodium acetate buffer at the pH of 5 and filtered with 0.45 μm syringe filters without other purification. To assay the activity of laccase in the presence/ absence of PAA-T, two types of laccase solution were prepared. One contained 67 mg/L laccase in 50 mM sodium acetate (pH 5), and the other with 67 mg/L laccase with 67 mg/L PAA-T25 also in acetate buffer. The samples were aged at room temperature in a 20 mL glass bottle fitted with a plastic cap without mixing. Periodically, 0.04 mL of laccase solution was removed from the beakers and the residual laccase activity was measured by the following method. Forty microliters of laccase solution was added to 3 mL of 0.5 mM ABTS in 50 mM sodium acetate buffer (pH 5) at 25 °C in a 1 cm quartz spectrophotometer cuvette. The absorbance at 420 nm (DU 800 Spectrophotometer) was measured over a period of 1.5 min, yielding a linear plot with time corresponding to the oxidation of ABTS. The slopes of the absorbance plots were converted to relative laccase activity, U = μmol ABTS/min, using the published molar extinction coefficient for the ABTS oxidation (ε420 = 36 000 M−1 cm−1). All measurements were performed in triplicate.16 Cellulose Membrane Oxidation and PVAm Grafting. Cleaned dialysis tubing was cut along the lengthwise direction to form either 2 × 6 cm “top membranes” or 3 × 6 cm “bottom membranes” for the laminates. Our membrane oxidation and PVAm grafting with PAA-T mediated laccase/O2 oxidation involves three steps: (1) four pairs of cellulose membranes were immersed in the 1.0 g/L PVAm solution for 30 min to give a saturated adsorbed monolayer of PVAm on the membranes, followed by rinsing with sodium acetate (50 mM, pH 5) 5 times to remove excess unadsorbed PVAm; (2) the cellulose membranes were immersed in a PAA-T solution, (20 mg of PAA-TEMPO in 130 mL of sodium acetate buffer) (50 mM,
Figure 1. Cellulose oxidation/PVAm grafting procedures for three mediators (TEMPO, PVAm-T, and PAA-T) and two primary oxidants (NaClO/NaBr or laccase/O2).
Regenerated cellulose dialysis membrane was purchased from Spectrum Laboratories (Rancho Dominguez, CA). A molecular weight cut-off (MWCO) of 6000−8000 (Spectra/Pro 1) was used for dialysis, and a MWCO of 12 000−14 000 (Spectra/Pro 2) was used for laminate sample preparation. Both were boiled for 30 min and washed 3 times with deionized water to remove additives before use. 4-Amino-2,2,6,6-tetramethyl-1-piperidinyloxy (4-aminoTEMPO), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and laccase from Trametes versicolor (EC 1.10.3.2) were purchased from Sigma-Aldrich. Other salts for buffer preparation and acid or base for pH adjustment were purchased from Caledon Laboratories. All solutions were made with Type 1 water (18.2 MΩ cm−1, Barnstead Nanopure Diamond system). PAA-T Preparation. EDC chemistry was used to condense 4-amino-TEMPO with poly(acrylic acid) and the recipes are given in Table 1. Table 1. Grafting Conditions and Resulting PAA-TEMPO Compositions
designation
PAA (g)
4-aminoTEMPO (g)
EDC (g)
grafting degree (%)
TEMPO content (wt %)
PAA-T5 PAA-T13 PAA-T25 PAA-T39
2.0 2.0 2.0 2.0
0.08 0.21 0.40 0.65
0.4 1.0 2.0 3.2
4.9 12.9 24.7 38.6
9.8 23.2 38.6 52.3
PAA-T12
0.5
0.05
0.24
12.4
22.4
appearance white white white light yellow white 4749
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laminate samples were attached to a freely rotating aluminum wheel1 with double-side tape (3 M polyethylene medical double coated tape, Model No: 1522). The delamination forces (90° peeling) were measured with a 50 N load cell on an Instron 4411 universal testing system at a strain rate of 20 mm/ min. The delamination force was an average value of steadystate peeling loads divided by the width of the interaction area (20 mm). Usually, four laminate samples were tested for each sample in order to get the average delamination force and the standard error. Adsorption on Cellulose QCM-D Sensors. The adsorption of various sequences of PVAm, PAA-T, and laccase on cellulose was measured with a Q-sense quartz crystal microbalance (E4 module from Q-sense, Gothenburg, Sweden) fitted with QSX 334 cellulose sensors. All solutions were prepared in sodium acetate buffer (50 mM pH 5). The sensors were conditioned with buffer at a flow rate of 0.25 mL/min. The polymer solutions 0.1 g/L PVAm, 0.1 g/L PAA-T25, or 0.1 g/L laccase were added at a flow rate of 0.15 mL/min at 25 °C.
pH 5), for 30 min; (3) 20 mL of 1.0 g/L laccase solution in sodium acetate (50 mM, pH 5) filtered with a syringe filter (0.45 μm) was added into the suspension of cellulose films in PAA-TEMPO solution. The resulting mixture was stirred under oxygen purging to initiate the oxidation at room temperature. The oxidation time was varied. The resulting oxidized cellulose membranes were rinsed in sodium acetate buffer (50 mM, pH 5) five times. Fluorescent Labeling of Aldehydes. Fluorescent labeling was used to indicate the presence of aldehyde groups on oxidized cellulose. The membranes were immersed in 0.05 g/L fluorescein-5-thiosemicarbazide in phosphate buffer overnight at pH 8. The cellulose membranes were rinsed with pH 8 PBS buffer 10 times to remove the excess fluorescein probe. The three membranes were stacked together and sectioned wet with a Leica CM3050S cryostat. Images of the sections were obtained with a ZEISS 510 inverted confocal microscope using an excitation wavelength of 488 nm. Layer-by-Layer (LbL) Membrane Treatment. For some experiments, polymer multilayers were formed on the cellulose films using LbL assembly. PVAm, PAA-T, and laccase solution were all prepared in sodium acetate buffer (50 mM, pH 5). Four pairs of cellulose membranes were soaked in 1.0 g/L PVAm solution for 30 min first and then rinsed with sodium acetate (50 mM, pH 5) five times to remove the excess unadsorbed PVAm. After removal of the buffer on the membrane surface, the cellulose membranes were dipped in 1.0 g/L PAA-T solution for 30 min, followed by rinsing with buffer. Then the cellulose membranes were dipped in 1.0 g/L laccase solution for 30 min and rinsed five times with buffer solution. The resulting generation 1 membranes were designated LbL 1. At this point, the membranes were either allowed to oxidize in buffer with an oxygen purge or were further treated with another generation of PVAm + PAA-T + laccase, giving the second generation of multilayers LbL 2. Membranes with three generations, LbL 3 were also prepared and allowed to oxidize in buffer with an oxygen purge at room temperature for 24 h. Lamination and Delamination Force Measurements. Pairs of oxidized cellulose membranes were laminated together using a thin (15 mg/m2) PVAm layer. The bottom and top oxidized cellulose membranes were placed on a polished TAPPI standard stainless-steel plate. Excess buffer on the membrane surface was gently blotted with Kimwipes tissues. A strip of nonadhesive Teflon tape (40 × 12.7 mm, G. F. Thompson, TWV480P) was placed across one end of the bottom membrane. The Teflon tape served as a release layer facilitating the separation the bottom and top membranes when the end of the top membrane is attached to the jaws of the testing machine. Fifteen microliters of 1.0 g/L PVAm adhesive solution was deposited along the center of the bottom membrane. After which, the top membrane was gradually placed over the center bottom membrane, spreading PVAm adhesive, uniformly across the laminate. Finally, the laminate was pressed between two TAPPI standard blotters in a Carver press for 30 min with 6000 pounds of force at room temperature. The resulting laminates were dried overnight at 23 °C and 50% relative humidity. Before testing, the laminates samples were soaked in Tris− HCl buffer (3 mmol/L, pH 7.5) for 30 min. The wet laminate samples were taken from the buffer and placed between two pieces of blotting paper. A 2.4 kg stainless steel roller was then used to press the laminate sample to remove excess buffer. The
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RESULTS Polymer-T Preparation and Properties. Figure 1 shows the structures of three oxidation mediators, TEMPO, PVAm-T, and PAA-T. PVAm-T preparation was described previously,12 whereas this work is the first report of PAA-T synthesis and use as an oxidation mediator. In both cases, synthesis involved the EDC mediated condensation. Reaction conditions and the resulting PAA-T compositions are summarized in Table 1. The TEMPO contents ranged from 4.9 to 39% of substitution of the carboxyl groups. PAA-T39, with the highest TEMPO content, had limited water solubility and a light yellow color. Most of the experiments were performed with PAA-T25 at pH 5 in 50 mM acetate buffer. Under these conditions, PAA-T25 had a negative charge content, based on polyelectrolyte titration, of −1.6 meq/ g, which corresponds to a degree of ionization of 27% for the carboxyl groups. For comparison, the net charge content of our laccase was −1.34 meq/g (polyelectrolyte titration) and for PVAm, +10.7 meq/g.17,18 Not only are PVAm-T and PAA-T oppositely charged but PVAm-T has about an order of magnitude higher charge content at pH 5. Adsorption onto Cellulose. QCM-D was used to measure the adsorption of PAA-T, laccase, and PVAm onto cellulose and the results are summarized in Table 2. Frequency shifts were converted to coverages (mass/area) with the Sauerbrey model, as the dissipation values were low.19 Laccase adsorption onto untreated cellulose gave the highest coverage of all the combinations in Table 2, followed by laccase adsorption onto Table 2. Summary of QCM-D Adsorption Measurements Polymers and Laccase in 50 mM Acetate Buffer at pH 5 Measured by QCM-D experiment PVAm on cellulose PAA-T25 on PVAm coated cellulose laccase on PAA-T25 coated cellulose PAA-T25 on cellulose laccase on PVAm coated cellulose laccase on cellulose 4750
Δf 3/3 (Hz)
dissipation ΔD (×10−6)
Sauerbrey coverage (mg/m2)
−5.10 −14.5
0.49 0.55
0.90 2.6
−13.0
1.33
2.3
−4.40 −19.0
1.01 0.75
0.78 3.4
−21.1
1.88
3.7
dx.doi.org/10.1021/ie500280e | Ind. Eng. Chem. Res. 2014, 53, 4748−4754
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PVAm adsorbed layer becomes the surface primer layer. After the PVAm coated cellulose was washed, it was immersed in PAA-T solution followed by laccase/O2 treatment. The requirement for each step in Method 4/Figure 1 was demonstrated by the results of a series of experiments summarized in Table 3. The base case experiment involved all of the steps in Method 4 and gave a delamination force of 30 N/m; this is a high force. Indeed, forces above 40 N/m often cause tensile failure of the cellulose membranes. The remaining experiments summarized in Table 3 involve missing one of the components of the base case and for experiments 2−8 the adhesion was low. Two of the cases are significant. Experiment 5 with denatured laccase or experiments 3 and 7 with no laccase, gave poor adhesion. This is indirect evidence for the importance of cellulose oxidation. Experiment 4 in Table 3 shows that without the initial PVAm, adhesion was low. By contrast, NaClO/NaBr/TEMPO oxidation (Method 4 in Figure 4), gave high adhesion values without the presence of a PVAm layer before oxidation.1 One explanation is that insufficient anionic PAA-T adsorbs onto negatively charged cellulose surfaces to generate aldehyde groups. This hypothesis was tested by probing for the presence of aldehyde/hemiacetal groups on the cellulose films before application of the adhesive layer. Cellulose membranes were treated with aldehyde reactive fluorescein-5-thiosemicarbazide and then examined for fluorescence by confocal microscopy. Figure 3 compares the images of three membrane cross
PVAm coated cellulose. PAA-T25 on cellulose gave the lowest coverage. Finally, the Sauerbrey coverage of PVAm was 0.9 mg/ m2. Every combination tested resulted in some adsorption, including that of anionic PAA-T on cellulose. Interactions in Solution. Laccase, PVAm, and the grafted TEMPO polymers are all polyelectrolytes. Our previous work confirmed the expected polyelectrolyte complex formation between anionic laccase and cationic PVAm-T.20 These colloidal complexes were either cationic or anionic depending upon the mixing ratios. By contrast, we found no evidence of colloidal complexes between PAA-T and laccase in 50 mM acetate buffer. One of the consequences of polymer complexation with laccase is that the enzyme function can be decreased either by active site blocking or by protein denaturation. Figure 2 compares the decrease of laccase activity with time in buffer
Figure 2. Influence of PVAm-T and PAA-T on the catalytic activity of laccase as functions of the aging time in acetate buffer at room temperature.
versus in PVAm-T11 and in PAA-T25. Laccase activity half-life was about 9 h in cationic PVAm-T11 compared to around 200 h in PAA-T25. Indeed, laccase activity in PAA-T25 was similar to buffer alone, suggesting only weak interactions between laccase and PAA-T25. PAA-T as Cellulose Oxidation Mediator. Adhesion measurements were preformed as a sensitive but indirect assay for oxidation subsequent to PVAm grafting to regenerated cellulose. In spite of efforts to develop a simplified procedure, a multistep cellulose treatment was required to achieve high adhesion values. Figure 1 compares the treatment steps of PAAT mediated laccase oxidation (Method 4) to TEMPO and PVAm-T mediated oxidations with bleach or laccase. The first step in Method 4 is the adsorption of a layer of PVAm on the cellulose surfaces. After oxidized cellulose is grafted,2 this
Figure 3. Fluorescence microscope image of the cross section of a stack of three 160 μm thick cellulose membranes prepared by experiment No. 1, 4, or 7 in Table 3 except that the membranes were treated with an aldehyde-reactive fluorscent label instead of the PVAm adhesive. Only the bottom green membrane contained aldehyde groups.
sections. Only the bottom membrane with the adsorbed layer of PVAm was fluorescent. Furthermore, the surface regions displayed the highest fluorescent intensity reflecting the inability of the PAA-T to penetrate the cellulose film. In principle, the amine groups of PVAm, next to the oxidized cellulose surfaces, could have consumed all of the aldehydes.
Table 3. Influence of Cellulose Oxidation Conditions on the Wet Delamination Force for 24 h Oxidation Time in 50 mM Acetate Buffer at pH 5 No.
description
PVAm primer
PAA-T12 (mg/L)
laccase (mg/L)
PVAm adhesive (mg/m2)
1 2 3 4 5 6 7 8
base case only adhesive no oxidation no PVAm primer denatured laccase no PAA-T no laccase no adhesive
√ − √ − √ √ √ √
133 0 0 133 133 0 133 133
133 0 0 133 133 133 0 133
15 15 15 15 15 15 15 0
4751
delamination force (N/m) 30 3.3 5.0 6.0 9.3 6.2 8.1 6.9
± ± ± ± ± ± ± ±
3.4 0.4 1.0 1.3 1.5 0.2 0.4 0.8
dx.doi.org/10.1021/ie500280e | Ind. Eng. Chem. Res. 2014, 53, 4748−4754
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However, the images in Figure 3 show significant fluorescence, indicating that PVAm did not protect all of the aldehydes. Layer-by-layer Assembly. From a scientific/mechanistic perspective, the base case procedure in Table 3 (i.e., Method 4 in Figure 1) is poorly defined because of the sequential addition of PAA-T and laccase without a washing step. With a view to obtaining better defined laminate structures, PVAm primer, PAA-T mediator and laccase oxidant were assembled on cellulose membranes by layer-by-layer (LbL) assembly with intermediate washing steps. The QCM-D results in Table 2 suggest that the layers should form spontaneously. Three sets of LbL samples were prepared with 1, 2, or 3 sets of PVAm/ PAA-T/laccase layers. The coated cellulose membranes were soaked in oxygenated buffer for 24 h followed by lamination with PVAm adhesive. The adhesion results, summarized in Figure 4 show that LbL laminates were weaker than the “Base
Figure 5. Influence of TEMPO substitution on PVAm-T and PAA-T. The concentrations of PAA-T and laccase concentrations were 133 mg/L in 50 mM sodium acetate buffer at pH 5, and the oxidation time was 1 h.
both as an oxidant and a grafted primer layer. By contrast, anionic PAA-T does not form colloidal complexes with laccase. Instead, anionic PAA-T adsorbs on cellulose pretreated with cationic PVAm. The differences in these two systems are more clearly seen when comparing the effects of mediator and laccase concentrations. Figure 6 compares PVAm-T with PAA-T with respect to the laccase concentration in the oxidation step. PAA-T gave a
Figure 4. Comparing LbL laminates with those prepared by the Base Case method described in Table 3 and Figure 1. All measurements were made with PAA-T13 and all solutions were in 50 mM acetate buffer.
Case” laminates. Previously, we showed that LbL laminates based on PVAm-T were also weaker and we speculated that TEMPO moieties in LbL structures did not have sufficient mobility to shuttle electrons between laccase and the cellulose.20 Comparison of Oxidation Mediators. Over the past decade, we have worked with three oxidation mediators (TEMPO, PVAm-T, and PAA-T) with two primary oxidants (NaClO/NaBr and laccase/O2) as routes for grafting PVAm primer layers onto cellulose; these are now compared. The density of grafted TEMPO groups is an important structural variable for polymer-supported mediators and Figure 5 compares the new PAA-T mediator with PVAm-T. Detailed quantitative comparison of the various approaches is not valid because none of these experimental sets was optimized to give the maximum adhesion. Instead, these and the following results are compared to illustrate how adhesion with various mediators changes with the experimental variables. With laccase, anionic PAA-T gave approximately the same results as PVAm-T. However, the reader is reminded that the PAA-T laminate fabrication was slightly more complex than those with PVAm-T (see Figure 1). From a mechanistic perspective, it is surprising that the two mediators gave such similar results because their physical states are so different. Specifically, we showed previously that PVAm-T forms colloidal sized polyelectrolyte complex aggregates with laccase that serve
Figure 6. Influence of laccase concentration of wet adhesion with PAA-T and PVAm-T. Oxidized strips were laminated with 15 mg/m2 PVAm (45 kDa).
monotonic increase in adhesion with laccase concentration, suggesting that adhesion is sensitive to the oxidation kinetics. By contrast, PVAm-T gave a bell-shaped curve because with high laccase/PVAm-T ratios, the colloid complexes are negatively charged and tend not to adsorb onto cellulose.20 Figure 7 compares three mediators and two primary oxidants in terms of the influence of oxidation time on delamination force. PAA-T and PVAm-T with laccase showed similar results requiring the longest oxidations times. The classical TEMPO oxidation was the fastest; however, the interior of the cellulose film was degraded, ultimately giving film failure.
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DISCUSSION An overall goal of our work was to develop new approaches to graft a PVAm layer onto cellulose, “priming” the surface to expand the potential applications of cellulosic materials. Herein we have considered two primary oxidants and three TEMPO4752
dx.doi.org/10.1021/ie500280e | Ind. Eng. Chem. Res. 2014, 53, 4748−4754
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compared to free TEMPO because of the difficulty in transporting polymers across biological membranes. Which is superior, PVAm-T or PAA-T? Comparing Methods 3 and 4 in Figure 1 shows that PVAm-T involves fewer steps and results in cellulose surfaces coated with fewer components (PVAm-T + laccase versus PVAm + PAA-T + laccase). On the other hand, PAA-T does not form polyelectrolyte complexes with laccase whereas PVAm-T does, resulting in deactivation and precipitation in the solution phase (see Figure 2). To summarize, choosing the “best” PVAm grafting strategy from the methods in Figure 1 is complex. Table 4. Comparison of TEMPO-Based Oxidation Procedures
Figure 7. Influence of oxidation time on wet adhesion of cellulose membranes. The PAA-T25 and laccase treatment solution concentrations were 133 mg/L in 50 mM acetate buffer. Oxidized strips were laminated with 15 mg/m2 PVAm (45 kDa).
criterion lowest total TEMPO consumption cellulose bearing only grafted PVAm mildest conditions possibility of eliminating washing steps least number of steps
based oxidation mediators. Figure 1 summarizes the four main oxidation/grafting approaches that we have investigated. All four methods generated cellulose surfaces bearing at least a grafted monolayer of PVAm or PVAm-T. In addition to grafted PVAm or PVAm-T, Methods 3 and 4 in Figure 1 produce surfaces supporting other components. In Method 3, the cellulose surface bears both PVAm-T and laccase (before adhesive application and lamination) whereas in Method 4, the cellulose surface is covered with a combination of PVAm, PAAT, and laccase. Indeed, our previous work with Method 3 showed that the immobilized laccase retained some catalytic activity for a while.20 An obvious question from a technological perspective is which method in Figure 1 is the best. In terms of wet adhesion, optimized versions of all four methods give adhesion values approaching the cohesive strength of the supporting films. Consider first the primary oxidants. Bleach (NaClO) is inexpensive, gives the fastest oxidation, and involves the fewest steps of the four methods in Figure 1. On the negative side, bleach is a strong oxidant that degrades most organic materials and it requires pH 10−11, which is too high for some processes such as conventional papermaking. By contrast, laccase functions at moderate pH and is a mild oxidant. On the negative side, laccase is expensive compared to bleach, laccase requires longer oxidation times, and the resulting cellulose surfaces are contaminated by laccase. Considering the three TEMPO-based mediators, the choices are equally complex. TEMPO mediated oxidations are very popular in the scientific literature; however, there are real challenges in using TEMPO in commercial processes including expense, potential environmental impacts11 and the separation/concentration of TEMPO from aqueous process streams. By contrast, PVAm-T or PAA-T can be quantitatively retained on the cellulose surface, concentrating the TEMPO moieties where they are needed. This partitioning of TEMPO moieties on the cellulose surface means that less TEMPO is needed compared to free TEMPO in solution. In the case of papermaking where cellulose fibers are present in dilute suspension, we have estimated that by concentrating TEMPO moieties next to the cellulose surface, the overall TEMPO loading can be reduced by a factor of 1000.12 There are other benefits to immobilized TEMPO. Lignocellulosic surfaces treated with TEMPO show reduced rates of light induced yellowing.21 In addition, polymersupported TEMPO should have a lower biological impact
selective oxidation of cationic surfaces TEMPO concentrated on product, not in process streams
best method (Figure 1) 2 1 3, 4 3, 4 1, 3 (minus washing step) 4 2,3,4
PAA-T and PVAm-T both required at least 10% substitution to achieve high adhesion when using laccase (see Figure 5) whereas only 1%, the lowest tested, was needed for PVAm-T in the presence of bleach. In the case of PVAm-T, we proposed that a high grafting density was required to facilitate TEMPO moiety-to-TEMPO moiety electron transfer because individual immobilized TEMPO moieties had insufficient mobility to shuttle between the active site of laccase to the cellulose surfaces. Presumably, the same explanation holds for PAA-T. Electrochemical experiments currently underway in our laboratory may yield some support for this mechanism. Finally, to put this work in perspective, we acknowledge cellulose surface modification is not new. The paper industry routinely employs sizing agents and wet strength resins. Sizing agents are small hydrophobic molecules that covalently couple to cellulose,22 and wet strength resins are heat-activated crosslinking polymers that strengthen bonds between wet cellulose fibers.23 Similarly in the composites literature, a growing body of literature describes cellulose reactive polymers and oligomers designed to make cellulose compatible with plastic matrices.24 The academic literature contains many examples of polymer grafting from and grafting to cellulose. However, our focus has been on technologies involving cellulose modification under mild conditions in aqueous media, implementable in papermaking and related technologies. Two technologies closest in nature to our work are the heat activated irreversible deposition of carboxymethyl cellulose (CMC) onto cellulose,25,26 and the use of enzymes (XET) to graft a variety of materials into high molecular weight xyloglucan, a natural polymer that irreversibly adheres to cellulose.27 All three methods (CMC, XET, and our PVAm grafting) are technology platforms giving “primed” cellulose surfaces that can be further derivatized; the best one is application dependent.
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CONCLUSIONS (1) Surface grafting PVAm expands the property space of cellulose. However, when comparing three oxidation mediators 4753
dx.doi.org/10.1021/ie500280e | Ind. Eng. Chem. Res. 2014, 53, 4748−4754
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(11) Haseloff, R. F.; Mertsch, K.; Rohde, E.; Baeger, I.; Grigor’ev, I. A.; Blasig, I. E. Cytotoxicity of Spin Trapping Compounds. FEBS Lett. 1997, 418, 73−75. (12) Pelton, R.; Ren, P. R.; Liu, J.; Mijolovic, D. PolyvinylamineGraft-Tempo Adsorbs onto, Oxidizes and Covalently Bonds to Wet Cellulose. Biomacromolecules 2011, 12, 942−948. (13) Liu, J.; Pelton, R. Reactive Polyvinylamine-Graft-Tempo/ Laccase Complex Giving Wet Cellulose Adhesion. In Advances in Pulp and Paper Research: Transactions of the 14th Fundamental Research Symposium, SJ, I. A., Ed.; Pulp & Paper Fundamental Research Society: Cambridge, U.K., 2013; Vol. 2, pp 869−885. (14) Pelton, R.; Cabane, B.; Cui, Y.; Ketelson, H. Shapes of Polyelectrolyte Titration Curves. 1. Well-Behaved Strong Polyelectrolytes. Anal. Chem. 2007, 79, 8114−8117. (15) Tanaka, H. Effects of Salts on Colloid Titration. Jpn. TAPPI J. 1983, 37, 939−948. (16) Johannes, C.; Majcherczyk, A. Laccase Activity Tests and Laccase Inhibitors. J. Biotechnol. 2000, 78, 193−199. (17) Katchalsky, A.; Mazur, J.; Spitnik, P. Polybase Properties of Poly(Vinylamine). J. Polym. Sci. 1957, 23, 513−30. (18) Feng, X.; Pelton, R.; Leduc, M.; Champ, S. Colloidal Complexes from Poly(Vinyl Amine) and Carboxymethyl Cellulose Mixtures. Langmuir 2007, 23, 2970−2976. (19) Vogt, B. D.; Lin, E. K.; Wu, W.-L.; White, C. C. Effect of Film Thickness on the Validity of the Sauerbrey Equation for Hydrated Polyelectrolyte Films. J. Phys. Chem. B 2004, 108, 12685−12690. (20) Liu, J.; Pelton, R.; Obermeyer, J. M.; Esser, A. Laccase Complex with Polyvinylamine Bearing Grafted Tempo Is a Cellulose Adhesion Primer. Biomacromolecules 2013, 14, 2953−2960. (21) Mcgarry, P.; Heitner, C.; Schmidt, J.; Seltzer, R.; Cunkle, G.; Wolf, J. P. Hindered Nitroxide: A New Yellowing Inhibitor for Mechanical Pulps. J. Pulp Paper Sci. 2000, 26, 59−66. (22) Lindstrom, T.; Larsson, P. T. Alkyl Ketene Dimer (Akd) Sizing a Review. Nord. Pulp Pap. Res. J. 2008, 23, 202−209. (23) Espy, H. H. The Mechanism of Wet-Strength Development in Paper: A Review. Jpn. TAPPI J. 1995, 78, 90−99. (24) Hubbe, M.; Rojas, O.; Lucia, L.; Sain, M. Cellulosic Nanocomposites: A Review. BioResources 2008, 3, 929−980. (25) Laine, J.; Lindstrom, T.; Nordmark, G. G.; Risinger, G. Studies on Topochemical Modification of Cellulosic Fibres Part 1. Chemical Conditions for the Attachment of Carboxymethyl Cellulose onto Fibres. Nord. Pulp Pap. Res. J. 2000, 15, 520−526. (26) Orelma, H.; Teerinen, T.; Johansson, L.-S.; Holappa, S.; Laine, J. Cmc-Modified Cellulose Biointerface for Antibody Conjugation. Biomacromolecules 2012, 13, 1051−1058. (27) Zhou, Q.; Rutland, M. W.; Teeri, T. T.; Brumer, H. Xyloglucan in Cellulose Modification. Cellulose 2007, 14, 625−641.
(TEMPO, PVAm-g-TEMPO, and PAA-g-TEMPO) and two primary oxidants (bleach and laccase), there is no “best” approach to grafting PVAm onto cellulose; each combination has strengths and compromises. (2) Only bleach/TEMPO gives a cellulose surface bearing only grafted PVAm whereas PVAm-T, PAA-T, and laccase give cellulose surfaces with immobilized TEMPO moieties and active laccase. (3) Unlike cationic PVAm-T, anionic PAA-T does not flocculate laccase in solution suggesting PAA-T may be more robust in many applications. (4) PAA-T requires an adsorbed layer of PVAm on cellulose to promote PAA-T adsorption and oxidation. PAAT + laccase does not oxidize untreated cellulose. (5) PAA-T and PVAm-T require more than 10% TEMPO substitution when used with laccase; we propose that this high TEMPO density is required for TEMPO-to-TEMPO electron transport along the PAA-T chains when laccase is the primary oxidant.
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AUTHOR INFORMATION
Corresponding Author
*R. Pelton. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Shuxian Shi (File No. 2011811369) acknowledges the China Scholarship Council for supporting her tenure in Canada as a visiting scholar. BASF Canada and NSERC are acknowledged for funding R.P. and Q.F. Pelton holds the Canada Research Chair in Interfacial Technologies.
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REFERENCES
(1) Diflavio, J. L.; Bertoia, R.; Pelton, R.; Leduc, M., The Mechanism of Polyvinylamine Wet-Strengthening. In Advances in Paper Science and Technology: Transactions of the 13th Fundamental Research Symposium, SJ, I. A., Ed.; Pulp & Paper Fundamental Research Society: Cambridge, U.K., 2005; Vol. 1, pp 1293−1316. (2) Diflavio, J. L.; Pelton, R.; Leduc, M.; Champ, S.; Essig, M.; Frechen, T. The Role of Mild Tempo-Nabr-Naclo Oxidation on the Wet Adhesion of Regenerated Cellulose Membranes with Polyvinylamine. Cellulose 2007, 14, 257−268. (3) Kitaoka, T.; Isogai, A.; Onabe, F. Chemical Modification of Pulp Fibers by Tempo-Mediated Oxidation. Nord. Pulp Pap. Res. J. 1999, 14, 279−284. (4) Anelli, P. L.; Biffi, C.; Montanari, F.; Quici, S. Fast and Selective Oxidation of Primary Alcohols to Aldehydes or to Carboxylic-Acids and of Secondary Alcohols to Ketones Mediated by Oxoammonium Salts under 2-Phase Conditions. J. Org. Chem. 1987, 52, 2559−2562. (5) Fabbrini, M.; Galli, C.; Gentili, P.; Macchitella, D. An Oxidation of Alcohols by Oxygen with the Enzyme Laccase and Mediation by Tempo. Tetrahedron Lett. 2001, 42, 7551−7553. (6) Arends, I. W. C. E.; Li, Y.-X.; Ausan, R.; Sheldon, R. A. Comparison of Tempo and Its Derivatives as Mediators in Laccase Catalysed Oxidation of Alcohols. Tetrahedron 2006, 62, 6659−6665. (7) Kulys, J.; Vidziunaite, R. Kinetics of Laccase-Catalysed Tempo Oxidation. J. Mol. Catal. B: Enzym. 2005, 37, 79−83. (8) Barreca, A. M.; Sjögren, B.; Fabbrini, M.; Galli, C.; Gentili, P. Catalytic Efficiency of Some Mediators in Laccase-Catalyzed Alcohol Oxidation. Biocatalysis and Biotransformation 2004, 22, 105−112. (9) Lee, S.-K.; George, S. D.; Antholine, W. E.; Hedman, B.; Hodgson, K. O.; Solomon, E. I. Nature of the Intermediate Formed in the Reduction of O2 to H2O at the Trinuclear Copper Cluster Active Site in Native Laccase. J. Am. Chem. Soc. 2002, 124, 6180−6193. (10) Aracri, E.; Vidal, T.; Ragauskas, A. J. Wet Strength Development in Sisal Cellulose Fibers by Effect of a Laccase−Tempo Treatment. Carbohydr. Polym. 2011, 84, 1384−1390. 4754
dx.doi.org/10.1021/ie500280e | Ind. Eng. Chem. Res. 2014, 53, 4748−4754
