Effect of Water-Mediated Arylation Time on the Properties of Soy

Publication Date (Web): March 1, 2010 ... ISP and DSP protein films were arylated with 2,2-diphenyl-2-hydroxyethanoic acid, in the presence of water, ...
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Ind. Eng. Chem. Res. 2010, 49, 3479–3484

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Effect of Water-Mediated Arylation Time on the Properties of Soy Protein Films Rakesh Kumar* CSIR Materials Science and Manufacturing, P.O. Box 1124, Port Elizabeth 6000, South Africa

In situ and dip coating methods were used to prepare arylated soy protein films designated as ISP and DSP, respectively. ISP and DSP protein films were arylated with 2,2-diphenyl-2-hydroxyethanoic acid, in the presence of water, at different time intervals to impart the hydrophobicity in the protein films. DSP and ISP series films suffered a dramatic loss of mechanical properties with the increase in arylation time. Morphological characterization of the soy protein films arylated at different time intervals was done by scanning electron microscopy. It was observed that the increase in arylation time leads to the clustering of the hydrophobic units, through hydrophobic interactions, resulting in the hydrophobic collapse. Hydrophobic collapse in the arylated protein films showed significant decrease in the mechanical properties of the protein films. However, the water uptake of these protein films was independent of the arylation time. The immediate implications of these experimental results are related to the promising use of water-mediated arylated soy proteins as films and composites. 1. Introduction

2. Experimental Section

The low-cost and ecofriendly characteristics of protein-based “green” materials have attracted researchers.1,2 In recent years, films and composites based on protein polymers are prepared not only from natural protein-based materials,3–6 but also from polypeptides.7 To improve the water resistance and the mechanical properties of protein-based materials ethylene glycol,8 acetamide,9 polyurethane,10 and stearic acid11 had been used. Recently, we also reported use of 2,2-diphenyl-2-hydroxyethanoic acid (DPHEAc) as an amphiphilic additive to increase water resistance and mechanical properties of soy protein films.12,13 Interestingly, water was used as a medium to induce hydrophobicity in the soy protein films through the process of dipcoating or in situ arylation. It was established from our earlier research findings that arylated protein films show very low water uptake when immersed in water as compared to nonarylated ones.12,13

2.1. Materials. Soy proten isolate (SPI) with about 90.27% (dry basis) of a protein was purchased from Zhenghou Ruikang Enterprise Co., Ltd. (Zhengzhou, China). Thiodiglycol (TDG) (bp ) 164-166 °C, mol wt ) 122.19, and density ) 1.182 g/cm3) and DPHEAc (mp ) 149-151 °C, mol wt ) 228.25) were purchased from Sigma and used as received. 2.2. Preparation of Arylated SPI Films. TDG (30% w/w wrt to SPI) was mixed with SPI powder, separately, in an electronic mixer for about 15 min and used to prepare arylated SPI films using two approaches described by us earlier.12,13 In the first approach, plasticized soy protein mixtures were further mixed with 10% DPHEAc (% w/w wrt the SPI/TDG mixtures) and subjected to hot press at 155 °C for 10 min under 50 bar pressure. The arylated protein films were immersed in distilled water at room temperature for different time intervals, from 4 to 24 h, to get the water-induced hydrophobic arylated soy protein films. The in situ arylated soy protein films obtained after immersion in distilled water for 0, 4, 8, 12, 16, 20, and 24 h were represented as ISP-0, ISP-1, ISP-2, ISP-3, ISP-4, ISP5, and ISP-6, respectively. In the second approach, plasticized soy protein mixtures were subjected to hot press at 140 °C for 20 min under 50 bar pressure to obtain the films. DPHEAc solution was prepared by dissolving 0.5 g of DPHEAc in 100 mL of boiling water and then cooling it to room temperature under constant stirring to prevent the growth of the DPHEAc crystals. The soy protein films were immersed in DPHEAc solution (0.5% w/v) for 0, 4, 8, 12, 16, 20, and 24 h to get dip coated arylated protein films. Dip coated arylated protein films obtained after 0, 4, 8, 12, 16, 20, and 24 h of arylation were represented as DSP-0, DSP-1, DSP-2, DSP-3, DSP-4, DSP-5, and DSP-6, respectively. 2.3. Characterizations. The tensile strength (TS), elongation at break, and the Young’s modulus (E) of the DSP and ISP series protein films were measured on an Instron 3369 testing machine with a tensile rate of 10 mm min-1 according to ASTM D882 (E) after conditioning the films at 57% RH for 3 days. The cracks in few protein films obtained upon conditioning the protein films at 57% RH were evaluated by optical microscope (Olympus SZ61) at 40× magnification in transmission mode equipped with digital image processing software. Dynamic mechanical thermal analysis (DMTA) was performed on a

In 1959, it was reported that in biological structures an interactionmediatedbywatersthehydrophobicinteractionsseemed to cause clustering of hydrophobic units, and this idea is now widely accepted.14–16 In these systems, water acts as a solvent and is made to evaporate after completion of reaction to get the desired materials. Drying-induced hydrophobic polymer collapse17 and solvent induced ordering and disordering of the phenyl side branches at the air/polystyrene interface has been observed.18 In our recent research findings, we have shown water is being used as a medium to generate the hydrophobic diphenyl hydroxy methane (DPHM) from DPHEAc in soy protein films.12,13 In this communication, we have investigated the critical role of arylation time on the properties of the soy protein materials. Surface morphology was done to study the nature of the DPHM microparticles upon arylation at different time scales. In particular, these findings will have remarkable implications on the promising use of arylated soy protein films and composites where the mechanical properties in addition to the water resistance of the final products are very important. * To whom correspondence should be addressed. Tel.: +27-415083263. Fax: +27-41-5832325. E-mail address: krrakesh72@ gmail.com.

10.1021/ie901930g  2010 American Chemical Society Published on Web 03/01/2010

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Figure 1. Mechanical properties on the DSP (above) and ISP (bottom) series films. Photograph on the right-hand side shows the cracks being generated in the DSP-6 and ISP-6 protein films when conditioned at 57% RH for mechanical testing.

dynamic mechanical analyzer (DMA8000, Perkin-Elmer, U.S.A.) with dual cantilever at a frequency of 1 Hz. The 50 mm × 8 mm (length × width) films were heated at the rate of 2 °C per min between the temperature ranging from 20 to 240 °C, and the R-relaxation temperature, Rr, was determined as the peak value of the loss angle tangent (tan δ). Thermogravimetric analysis (TGA) of approximately 5 mg films was carried out at a heating rate of 10 °C min-1 between room temperature and 700 °C under a nitrogen atmosphere on a TG-IR interface (Perkin-Elmer, U.S.A.). Scanning electron microscopy (SEM) images of the surface of the arylated protein films were taken on an FEI Quanta 200 (Eindhoven, The Netherlands) electron microscope at an accelerating voltage of 5 kV in low vacuum to prevent the damage in the samples. Fourier transform infrared spectra (FTIR) of the DSP and ISP series protein films were carried out on a Spectrum 100 FTIR (Perkin-Elmer USA) in the range from 4000 to 600 cm-1 using the powdered samples. The fluorescence spectral property of the arylated protein films, dissolved in ethanol solution, has been studied on a photoluminescence spectrometer (Perkin-Elmer LS-55 Fluorescence Spectrometer, USA). A xenon lamp was used as an excitation source. Excitation and emission slit widths were 6 nm. The water uptake of the DSP and ISP series protein films was evaluated according to ASTM D570-81. The arylated protein films were preconditioned at 50 °C for 24 h and weighed

(W0). After immersion in distilled water, the films were dried with paper towels to remove the excess water on the surface and weighed (W1). The weight gain of the films counted as the total to calculate the absorbed water. An average value from three measurements was reported. The water uptake of the films was calculated as follows % water uptake )

w1 - w0 × 100 w0

The containers with water-soluble materials were also placed in an oven at 50 °C for 24 h to obtain the water-soluble contents. 3. Results and Discussions 3.1. Properties of the Films. The stress-strain curves of the ISP and DSP series of films conditioned at 57% RH for 3 days are shown in Figure 1. Interestingly, percentage elongation at break in the case of DSP series of protein films was higher than that for the ISP series films. The tensile strength and modulus of DSP series films exhibited a decrease in their values from 18.5 MPa (for DSP-1) to 8.5 MPa (for DSP-3) and 826 MPa (for DSP-1) to 166 MPa (for DSP-3), respectively, with the increase in the arylation time from 4 to 12 h. Interestingly, DSP series films crumbled after 16 h of arylation (shown in

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Figure 3. Water uptake of DSP and ISP series films. Table 1. Dependence of r-Relaxation Temperature on the Arylation Time DSP series films

Figure 2. Thermogravimetric curves of the protein films prepared by dip coating arylation (above) and in situ arylation (below).

top right-hand side of Figure 1) and broke into several pieces when conditioned at 57% RH for mechanical testing. Before discussing the mechanical properties of ISP series films, we would like to discuss apparent water uptake of ISP-0 when immersed in water for up to 24 h at different time intervals. With the increase in immersion time for ISP-0 from 2 to 10 h, there is decrease in the apparent water uptake from 32.0% to 21.6% (Supporting Information Figure SI-1). Interestingly, after 10 h of immersion of ISP-0 in water, apparent water uptake values remained constant. The decrease in apparent water uptake is due to the loss of carbondioxide from DPHEAc to form DPHM, and the reaction mechanism of this phenomena has already been reported in our previous paper.12,13 ISP-3 showed tensile strength and modulus of 11 and 881 MPa, respectively, whereas a tensile strength of 3.8 MPa and modulus of 186 MPa decreases for ISP-5 again showed that mechanical properties decrease with an increase in the arylation time. The ISP series films were very brittle and crumbled into pieces before 8 h and after 24 h of arylation (shown in the bottom right-hand side of Figure 1). It can be stated that with the increase in the arylation time for the ISP/DSP series protein films, mechanical properties showed a decrease in their values leading to a mechanical failure. The decrease in the mechanical properties at higher arylation time may be attributed to the inherent driving forces of the DPHM hydrophobic and soy protein hydrophilic units. There may be interfacial tension with hydrophilic soy protein units trying to absorb the moisture at 57% RH while the hydrophobic DPHM units are trying to suppress it. This

samples

R-relaxation temperature (°C)

DSP-0 DSP-1 DSP-2 DSP-3 DSP-4 DSP-5 DSP-6

138.1 196.0 197.3 197.5 197.5 198.7 200.7

ISP series films samples ISP-0 ISP-1 ISP-2 ISP-3 ISP-4 ISP-5 ISP-6

R-relaxation temperature (°C) 70.3 208.2 209.4 211.9 212.4 212.9 215.1

interfacial tension leads to the cracks in the protein films prepared at high arylation time. Figure 2 shows the thermogravimetric curves of the protein films arylated at different time scales. Mass loss of the arylated protein films was determined at 200 °C. DSP-0 showed the mass loss of 10% at 200 °C, while the arylated samples showed mass loss of 4-4.5%. This indicated less absorption of moisture by the arylated protein films. With the increase in the arylation time, there are increases in maximum degradation temperature (Tmax) from 335 °C for DSP-0 to 360 °C for DSP-5. For ISP-4, Tmax further increased to 368 °C. Additionally, there were also increases in the char yield at 700 °C, for the arylated soy protein films, with the increase in the arylation time. Overall, we can state that increase in the arylation time showed an increase in the thermal properties. The increase in thermal stabilities is attributed to the incorporation of aromatic backbone in soy protein.19 Figure 3 shows the time dependence of water uptake on the arylated protein films at different time intervals. For ISP-0, a very low value of water uptake (apparent water uptake) was due to the loss of carbon dioxide from the samples in addition to the absorbed water as discussed in our earlier paper.12 DSP-0 showed about 105% water uptake. The value of water uptake for DSP-0 is 25% higher than that for the TDG-plasticized soy protein films, reported by us earlier,20 and this could be attributed to the change in the soy protein as a raw material. DSP series protein films showed lower water uptake than that of ISP series films. The average soluble residue for all the DSP and ISP samples was found to vary between 0.17 and 0.51%. On the contrary, the average soluble residues for ISP-0 (∼2.5%) and DSP-0 (∼3.3%) was found to be higher than water-mediated arylated protein films. Our earlier research findings which state that the protein films/composites after arylation showed significant decrease in water uptake are well demonstrated here

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Figure 5. Fourier transforms infrared spectra of (a) ISP and (b) DSP films.

Figure 4. Surface morphology of DSP (left) and ISP (right) series films. The scale for ISP-5 and DSP-5 is 5 µm while for all other protein samples it is 10 µm.

also.12,13,21 Overall, we can conclude that the arylation of protein films reduces the water uptake of about 50-55% and that it is independent of the arylation time. Table 1 shows the R-relaxation temperatures for the protein films arylated at different time scales. The R-relaxation temperatures for DSP-0 (138.1) was higher than ISP-0 (70.3) indicating the decrease in the molecular mobility of the protein molecules with the addition of DPHEAc in ISP-0 (Supporting Information Figure SI-2). Upon arylation, the R-relaxation temperatures increased again indicating less molecular mobility

of the protein chains, and this is due to the introduction of aromatic backbone in soy protein molecules. It is also interesting to note that, upon arylation, ISP series protein films showed higher R-relaxation temperatures than that of DSP series protein films. 3.2. Surface Morphology of the Films. Figure 4 shows the surface morphology of the protein films arylated at different time scales. DSP-0 showed homogeneous surface while DSP-1 showed almost even distribution of DPHM microparticles obtained from DPHEAc by evolution of CO2. The increase in arylation time increased the number of DPHM microparticles on the surface of soy protein films as evident from the surface morphology of DSP-3. The surface morphology of DSP-5 showed the formation of DPHM cluster rather than DPHM microparticles. The appearance of the DPHM cluster is due to hydrophobic interactions among the DPHM microparticles. ISP series protein films were prepared by incorporation of DPHEAc, so ISP-0 showed the presence of DPHEAc particles on the surface. With the increase in the arylation time, the number of DPHEAc particles decreased and the formation of DPHM microparticles increased.12 ISP-3 showed the presence of evenly distributed DPHM microparticles. The increase in the arylation time started to favor hydrophobic interactions in DPHM microparticles as evidenced by the formation of few clusters for ISP-5. It has been observed in our system that hydrophobic interactions among DPHM increase in strength with increasing

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Figure 6. Liquid solution fluorescent emission spectra of (a) DSP and (b) ISP at room temperature. The solid line, dashed line, and dotted lines in Figure 6a indicate DSP-0, DSP-1 and DSP-4, respectively. Similarly, the solid line, dashed line, and dotted lines in Figure 6b indicate ISP-0, ISP-3, and ISP-6, respectively.

arylation time. DPHM hydrophobic units reduce the volume of configuration space available for hydrogen bonding with soy protein and form a cluster.22 Overall, it can be stated that the hydrophobic collapse is observed when hydrophobic units exist as clusters rather than particles.17 3.3. Structural Characterization. Figure 5 shows the FTIR spectra of the arylated protein films. A broad N-H stretching band at 3272-3275 cm-1 is assigned to amide A of the soy protein in DSP and ISP series films. The band at 1528 cm-1 in the DSP-0 sample, containing only soy protein, is attributed to the N-H bending and upon arylation this band shifted to the lower frequency due to aromatic C-C stretching confirming the introduction of aromatic backbone. The presence of a strong band at 698 cm-1 (out-of-plane aromatic C-H bending) in the spectra of the ISP and DSP series of samples, except that in DSP-0, again confirmed the introduction of aromatic backbone in soy protein. Figure 6 shows the fluorescent spectral property of the arylated protein films in ethanol solution. Protein films exhibited a narrow band with maxima at 358 nm which is attributed to the aryl groups.23 The spectral broadening of the band at 358 nm with the increase in arylation time reveals the clustering of the aryl groups.24 On the basis of the above observation, a scheme has been proposed in Figure 7. In the case of a DSP series film, during the initial hour of arylation there is hydrogen bond between

Figure 7. Schematic illustration of the interaction between SPI and DPHM in the (a) initial and (b) final hour of arylation. The green part of the figure on the right-hand side denotes SPI while the red circles (•) represent the DPHM units.

soy protein and DPHM units (Figure 7a).22 As the arylation progresses with time, the hydrophobic interactions among the DPHM start to build up, and after 12 h of arylation, these DPHM hydrophobic units exist as a cluster (Figure 7b) resulting in hydrophobic collapse with lower mechanical properties (Figure 1). In the ISP series of film, the formation of hydrogen bonds between DPHM and soy protein only starts after 10 h of arylation i.e., when the loss of carbon dioxide from DPHEAc ceases (Supporting Information Figure SI-1).12 Hence for ISP3, there is a hydrogen bond between soy protein and DPHM units (Figure 7a). With the increase in arylation time, the formation of DPHM cluster through hydrophobic interactions starts taking place (Figure 7b) with lower mechanical properties (Figure 1). 4. Conclusions The optimum arylation times for the ISP and DSP series of protein films were found to be 4 and 12 h, respectively. The

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increased arylation time leads to the decreased mechanical properties in the protein films which is attributed to the hydrophobic collapse owing to the transformation of hydrophobic units from microparticles to a cluster structure. Hydrophobic collapse resulted in cracks in the films leading to mechanical failure. However, water uptake of the protein films was independent of the arylation time. This work establishes the fact that to fabricate arylated protein films, it is important to prevent hydrophobic collapse by controlling the arylation time. Acknowledgment Many thanks to Prof. Rajesh Anandjiwala, CSIR MSM, South Africa, for his professional help and much more. I would also like to thank Dr Arjun Maity, CSIR MSM, South Africa, for helping me in carrying out fluorescence spectroscopy. Supporting Information Available: Apparent water uptake for ISP-0 films at different time intervals (Figure SI-I) and temperature dependence of loss peaks (tan δ) for the DSP (a) and ISP (b) films (Figure SI-2). This material is available free of charge via the Internet at http://pubs.acs.org. Literature Cited (1) Wu, Q.; Sakabe, H.; Isobe, S. Processing and properties of low cost corn gluten meal/wood fiber composite. Ind. Eng. Chem. Res. 2003, 42, 6765–6773. (2) Wu, Q.; Yoshino, T.; Zhang, H.; Sakabe, H.; Isobe, S. Chemical modification of zein by bifunctional polycaprolactone (PCL). Polymer 2003, 44, 3909–3919. (3) Woerdeman, D. L.; Veraverbeke, W. S.; Parnas, R. S.; Johnson, D.; Delcour, J. A.; Verpoest, I.; Plummer, C. J. G. Designing new materials from wheat protein. Biomacromolecules 2004, 5, 1262–1269. (4) Zhang, X.; Do, M. D.; Bilyk, A. Chemical modification of wheatprotein-based natural polymers: formation of polymer networks with alkoxysilanes to modify molecular motions and enhance the material performance. Biomacromolecules 2007, 8, 1881–1889. (5) Foulk, J. A.; Bunn, J. M. Properties of compression-molded, acetylated soy protein films. Ind. Crops Prod. 2001, 14, 11–22. (6) Wang, Y.; Cao, X.; Zhang, L. Effects of cellulose whiskers on properties of soy protein thermoplastics. Macromol. Biosci. 2006, 6, 524– 531. (7) Deming, T. J. Polypeptide materials: New synthetic methods and applications. AdV. Mater. 1997, 9, 299–311.

(8) Wu, Q.; Zhang, L. Properties and structure of soy protein isolateethylene glycol sheets obtained by compression molding. Ind. Eng. Chem. Res. 2001, 40, 1879–1883. (9) Liu, D.; Zhang, L. Structure and properties of soy protein plastics plasticized with acetamide. Macromol. Mater. Eng. 2006, 291, 820–828. (10) Chen, Y.; Zhang, L.; Du, L. Structure and properties of composites compression-molded from polyurethane prepolymer and various soy products. Ind. Eng. Chem. Res. 2003, 42, 6786–6794. (11) Lodha, P.; Netravali, A. N. Thermal and mechanical properties of environment-friendly ‘green’ plastics from stearic acid modified-soy protein isolate. Ind. Crops Prod. 2005, 21, 49–64. (12) Kumar, R.; Zhang, L. Effect of water on the hydrophobicity of soy protein materials containing 2,2-diphenyl 2-hydroxyethanoic acid. Biomacromolecules 2008, 9, 2430–2437. (13) Kumar, R.; Zhang, L. Soy protein films with the hydrophobic surface created through non-covalent interactions. Ind. Crops Prod. 2009, 29, 485–494. (14) Tanford, C. How protein chemists learned about the hydrophobic factor. Protein Sci. 1997, 6, 1358–1366. (15) Kauzmann, W. Some factors in the interpretation of protein denaturation. AdV. Protein Chem. 1959, 14, 1–63. (16) Tanford, C.; Reynolds, J. Nature’s Robots: A History of Proteins; Oxford Re Univ. Press: Oxford, 2001; Chapter 12. (17) TenWolde, P. R.; Chandler, D. Drying-induced hydrophobic polymer collapse. Proc. Natl. Acad. Sci. 2002, 99, 6539–6543. (18) Opdahl, A.; Somorjai, G. A. Solvent vapor induced ordering and disordering of phenyl side branches at the air/polystyrene interface studied by SFG. Langmuir 2002, 18, 9409–9412. (19) Kaneko, T.; Thi, T. H.; Shi, D. J.; Akashi, M. Environmentally degradable, high-performance thermoplastics from phenolic phytomonomers. Nat. Mater. 2006, 5, 966–970. (20) Kumar, R.; Zhang, L. Aligned ramie fiber reinforced arylated soy protein composites with improved properties. Compos. Sci. Technol. 2009, 69, 555–560. (21) Kumar, R.; Wang, L.; Zhang, L. Structure and mechanical properties of soy protein materials plasticized by thiodiglycol. J. Appl. Polym. Sci. 2009, 111, 970–977. (22) Chandler, D. Interfaces and the driving force of hydrophobic assembly. Nature 2005, 437, 646–647. (23) Qui, Y.; Wang, K.; Liu, Y.; Deng, H.; Sun, F.; Cai, Y. Synthesis, characterization and 1D helical chain crystal structure of [Cu(DBA)2(1,10phen)]n and [Cd(DBA)2(1,10-phen)2] (DBA ) benzilic acid). Inorg. Chim. Acta 2007, 360, 1819–1824. (24) Brown, R.; Lacombe, S.; Cardy, H. Interpretation of spectral broadening and clustering of a pyrene derivative adsorbed on silica gels. Microporous Mesoporous Mater. 2003, 59, 93–103.

ReceiVed for reView December 10, 2009 ReVised manuscript receiVed February 19, 2010 Accepted February 19, 2010 IE901930G