Study on Camellia Oleifera Protein based Wood Adhesive by Epoxy Resin and Its Crosslinking Mechanism

CHEN Sicheng, LIANG Jiankun, ZHANG Bengang, WU Zhigang＊,LEI Hong＊, LI Lifen, YANG Shoulu

(1. College of Forestry, Guizhou University, Guiyang 550025, China; 2. School of Architectural Engineering, Kaili University, Qiandongnan 556011, China; 3. Yunnan Provincial Key Laboratory of Wood Adhesives and Glued Products, Southwest Forestry University, Kunming 650224, China; 4. Guizhou Academy of Forestry, Guiyang 550005, China)

Abstract: Epoxy resin (EPR) was used to crosslink with Camellia oleifera Abel. protein to prepare wood adhesive, and the bonding performance and curing characteristics of which were mainly investigated, and the synthesis mechanism was also discussed by using model compounds. The experimental results show that EPR can significantly improve the bonding performance of Camellia oleifera Abel. protein-based adhesive, and the maximum of which reaches 0.72 MPa satisfies the strength requirement of Type II plywood in GB/T 17657-2013. After alkali treatment, the protein can more easily crosslink with EPR at low curing temperature, and the adhesive has high degree of crystallinity of curing products, high degree of crosslinking reaction, and high bonding strength. The reaction mechanism of EPR-modified Camellia oleifera Abel. protein adhesive can be divided into resinification phase and curing phase.

Key words: Camellia oleifera protein; epoxy resin; crosslinking; wood adhesive

1 Introduction

More and more attention has been paid to production of environmentally friendly wood adhesives using agricultural and forest resources. Protein[1-4],starch[5,6], tannin[7,8], lignin[9,10], and other biomass materials[11,12]are usually applied to synthesis of wood adhesives. Due to the limitation of the variety of tannin, the research and application of tannin-based adhesives are rare in China. The complexity, variability and heterogeneit of lignin lead to unstable properties of lignin-based adhesives. High preparation cost,poor fluidity and poor water resistance make starchbased adhesives unsuitable directly using for wood.So, more researches have involved protein adhesives,which, according to source of raw materials, can be divided into animal protein adhesives and plant protein adhesives, where the researches on plant protein adhesives have a late start, but they have been investigated mostly at present. The plant protein adhesives which have been most successfully used is soy protein-based adhesives, some of which have realized industrialized production[13-16]. Even so, soy protein belongs to the grain resource and if it was applied to preparation of adhesives in a large scale, it will influence human food safety, and meanwhile, this is not advocated by Food and Agriculture Organization of the United Nations.

Camellia oleifera Abel. is also calledCamellia oleifera Abel. tree.Camellia oleifera Abel. seeds are mainly used to extractCamellia oleifera Abeloil. The total contents of fatty acids and unsaturated fatty acids inCamellia oleifera Abel., which is rich in nutrition,rank top among all kinds of edible oils, reaching 85%-95%. Tea oil has always been one of healthy edible oils popularized by Food and Agricultural Organization as a priority and it is praised as “olive oil in the Orient”.During the extraction process ofCamellia oleifera Abel. oil, a large quantity ofCamellia oleifera Abel.oil cake byproducts will be unavoidably generated.As reported,Camellia oleifera Abel. oil cake contains many constituents[17,18]: crude protein 19%-25%,saccharides 35%-40%, crude fat 5%, crude fiber 6%,saponin 10%-15%, and so on. UsingCamellia oleifera Abel. oil cake which is rich in proteins to prepare wood adhesives can provide a green protein adhesive for man-made boards especially plywood and will be of great importance to relieving dependence on soy proteins, reducing production cost and improving market competitiveness of protein-based adhesives.

Wood adhesive was prepared withCamellia oleifera Abel. proteins in this paper. The novelties of this work are: Development of a new protein-based adhesive without influence on human food safety;Development ofCamellia oleifera Abel. protein-based adhesive without formaldehyde emission by taken as epoxy resin the crosslinking agent; Crosslinking mechanism was studied with the help of model compounds.

2 Experimental

2.1 Materials

The camellia oleifera protein was from Guizhou University, China. Ala-Gln (AG) with a purity of 99%was purchased from Sinopharm Chemical Reagent Co., Ltd., Beijing, China. Epoxy resin (EPR) was a commercial product with name of E-44, whose epoxide number was 0.41-0.47 and softening point was 12-20 °C. Poplar veneer with the dimensions 400 mm× 400 mm × 2 mm and 8% to 10% moisture content was purchased from Qunyou Wood Co., Ltd. (Linyi,China) for plywood preparation. Other chemicals such as epichlorohydrin (ECH), NaOH were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai,China).

2.2 Preparation of protein-based adhesive

In a three-neck round-bottom flask equipped with a mechanical stirrer, thermometer and condenser was charged with 320 parts water. 80 parts camellia oleifera protein was then charged to the rapidly stirring solution. The mixture was heated to 45 ℃ and 21.3 parts 30% NaOH was added and stirring for 30min. The mixture was cooled to room temperature in an ice bath.The control adhesive sample was prepared in a similar way to above procedure, with no NaOH addition, for comparison purposes.

2.3 Preparation of samples prepared with AG and ECH

In a three-neck round-bottom flask equipped with a mechanical stirrer, thermometer and condenser was charged with 40 g water. 1 g AG was then charged to the rapidly stirring solution. The mixture was heated to 45 ℃ and 1 mL ECH was added and stirring for 40 min. The pH was kept as 8-9. The mixture was cooled to room temperature in an ice bath. The gotten products were used to the test of NMR and ESI-MS.

2.4 Preparation of plywood and testing of the shear strength

The camellia oleifera protein adhesives were used to prepare three-layer plywood with dimensions of 400 mm × 400 mm × 6 mm. Veneers with a doublesided adhesive loading of 220 g/m2were rested at room temperature for 15-20 min. The assembled veneers were then exposed to single-layer hot press unit (XLB type) at Shanghai Rubber Machinery Plant, and pressed with a pressure of 1.0 MPa at 120 °C for 8 min to obtain a plywood panel.

Triplicates of three-layer plywood panel were cut into shear specimens with dimensions of 100 mm× 25 mm. The wet shear strength of the plywood specimens was tested according to Chinese National Standard (GB/T 17657-2013) after subjecting to 63±3℃ water. A mechanical testing machine (model WDS-50 kN) was used to determine the shear strength of the plywood specimens. The reported wet shear strength is the mean of 10 specimens.

2.5 Differential scanning calorimetry (DSC)

A Perkin-Elmer DSC spectrometer with type DSC 204F1 purchased from Germany was used for the analysis. The thermal run for each sample was performed at a heating rate of 15 K/min. The temperature range was 30-250 ℃ for each sample. The software used for DSC data treatment was PYRISTM Version 4.0.

2.6 X-Ray diffraction (XRD)

X-Ray Diffraction spectra were obtained on X-Ray Diffraction (D/max-2200, Rigaku, Tokyo,Japan) equipped with Ni-filtered Cu Kα1radiation (λ=1.5406 Å) at 40 kV and 120 mA. The scattering angle range was 5° - 90° at a scan rate of 6 °/min.

2.7 Electrospray ionization mass spectrometry (ESI-MS)

The samples were dissolved in chloroform to get a solution with a concentration of 10 μL/mL, that was 10 μL sample and 1 mL chloroform. The solution was then injected into the ESI source plus ion trap mass spectrometer via a syringe at a flow rate of 5 μg/s.Spectra were recorded in positive mode with ion energy of 0.3 eV and scan range of 0-1000 Da on a Waters Xevo TQ-S instrument.

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2.8 13C Nuclear magnetic resonance spectroscopy (13C-NMR)

400 μL sample liquid was dissolved in 200 μL dimethyl sulfoxide-d6(DMSO-d6). A liquid13C-NMR(zgig) spectrum was then got from a Bruker Avance spectrometer with a frequency of 600 MHz. The chemical shifts were calculated relative to the control(CH3)3Si(CH2)3SO3Na dissolved in DMSO-d6. The spectra were done at 39062.5 Hz for a number of transients of 500-800. All the tests were run with a relaxation delay of 6 s and the chemical shifts were accurate to 1 ppm.

2.9 Fourier transform infrared spectroscopy(FT-IR)

About 0.001 g of sample powder was mixed well with 1 g of KBr to prepare a pill. The pill was then subjected to fourier transform infrared spectroscopy(FT-IR; Varian 1000; USA) and the data was acquired within the range of 400-4 000 cm-1at 4 cm-1resolution using 32 scans.

3 Results and discussion

3.1 Bonding performance of Camellia oleifera protein-based adhesive

Camellia oleifera Abel. protein contains a large quantity of hydrophilic groups with poor water resistance. Therefore,Camellia oleifera Abel.proteinbased adhesive can reach a certain strength and water resistance only when it is used together with crosslinking agent. Thermal expansion and contraction of plywood will generate a great influence on the adhesive, soCamellia oleifera Abel. protein-based adhesive applicable to indoor plywood should have superior water resistance and wet bonding strength.Epoxide is an active curing agent of alkaline soybean protein. Fig.1 shows the water resistance and bonding strength ofCamellia oleifera Abel. protein-based adhesives modified with different amounts of EPR.

Fig.1 Bonding performance of Camellia oleifera protein based adhesive with EPR

As shown in Fig.1, theCamellia oleifera Abel.protein-based adhesive had no water resistance.TheCamellia oleifera Abel. protein-based adhesive modified with EPR had remarkably high bonding strength. As the amount increase of EPR, the bonding strength firstly increased and then declined. Under the amount of 9%, the bonding strength ofCamellia oleifera Abel. protein-based adhesive was the maximum, being 0.72 MPa, thus satisfying the strength requirement of Type II plywood in GB/T 17657-2013(≥0.70 MPa). EPR was of great viscosity, so after it was mixed with degradation protein ofCamellia oleifera Abel., the viscosity of adhesive increased while enhancing the bonding performance, so as to influence operation performance. Moreover, EPR is expense,so the greater the amount, the higher the cost will be.Therefore, the amount of EPR reduced as much as possible while the performance was not influenced, and 9% of additive amount of EPR was appropriate.

3.2 DSC analysis

Fig.2 shows the DSC curve ofCamellia oleifera Abel. protein-based adhesive under heating rate of 15 K/min.Camellia oleifera Abel. protein-based adhesive had an obvious endothermic peak nearby 115 ℃,indicating that the main thermal reaction was completed below 120 ℃ and that hot pressing temperature 120℃ was proper. The curing temperature of un-treatedCamellia oleifera Abel.protein-based adhesive, which went through EPR crosslinking modification, was 115.3 ℃, while that ofCamellia oleifera Abel.proteinbased adhesive, which went through EPR crosslinking modification and NaOH degradation, was 112.5 ℃,manifesting thatCamellia oleifera Abel. protein after alkali treatment could more easily crosslink with EPR.

Fig.2 DSC curves of Camellia oleifera protein: (a) Control sample; (b) Sample treated by NaOH

3.3 XRD analysis

Fig.3 shows the 2θand degree of crystallinity results ofCamellia oleifera Abel. protein-based adhesive. As shown in Fig. 3,Camellia oleifera Abel.protein-based adhesive had two crystalline regions at diffraction angles nearby 12° and 22°, which were corresponding to α-helix crystalline region andβ-folded crystalline region ofCamellia oleifera Abel. protein molecules, respectively[19].

Fig.3 XRD patterns of Camellia oleifera protein adhesive: (a)Control sample; (b) Sample treated by NaOH

The relative crystallinity of undegradedCamellia oleifera Abel. protein after EPR crosslinking modification was 32.19%, but that ofCamellia oleifera Abel. protein degraded through NaOH was 34.32%,indicating that the crosslinking degree of molecular chains of the latter was elevated. TheCamellia oleifera Abel. protein after alkali modification had loose structure with molecules presenting asymmetric increase, resistance to motion and the viscosity increase, and proteins could be degraded, dispersed and stretched. on the one hand, polar groups and nonpolar groups were exposed, active sites were generated, and sufficient crosslinking with EPR was generated, thus improving the bonding performance. on the other hand,the contact area with wood fiber could be enlarged,and the interaction between hydrophilic groups and hydrophobic groups could be enhanced, so as to improve the bonding performance.

3.4 Modification mechanism of Camellia oleifera protein adhesive by the reaction among model compounds

Camellia oleifera Abel. protein has complicated constituents, so it’s difficult to directly study the modification mechanism ofCamellia oleifera Abel.protein-based adhesive by EPR. Following the research idea of model compounds, epoxy chloropropane was taken as model compound of EPR and AG as model compound ofCamellia oleifera Abel.protein, the structural changes of the reaction between the two compounds were investigated, and the modification mechanism of EPR-modifiedCamellia oleifera Abel.protein-based adhesive was studied.

ECH will firstly go through ring-opening reaction with AG. Tension exists in epoxy compound molecules due to its epoxy three-ring structure, and as a result, epoxy groups can experience ring-opening reaction with some nucleophilic reagents. ECH is a kind of asymmetric epoxy compound, and the ringopening direction of epoxy groups, which is decided by acid-base property of the reaction, should have high selectivity. Under alkaline condition, ring-opening reaction of epoxy groups on ECH proceeds according to the SN2 mechanism, and it mainly occurs at the end with few substituent groups, namely carbon atoms with relatively small steric hindrance, because what is formed under alkaline condition is a strongly alkaline alkoxyl anion, which is relatively stable and can not be easily delocalized.

Fig.4 shows the ESI-MS results of ECH and AG reaction products under alkaline condition. AG contained two free amino groups and one secondary amino group, where the latter almost did not react with ECH due to low reaction temperature under the test conditions. ECH mainly experienced free amino groups on AG. Amino N-1 replacement mainly occurred under charge-to-mass ratios of 310 Da (M+H+), 332 Da (M+Na+), and 348 Da (M+K+), and amino N-2 replacement under 402 Da (M+H+), 424 Da (M+Na+),and 440 Da (M+K+), and 2 replacement at the time might be N-2 replacement on an amino group, or two amino groups of one AG molecule simultaneously went through N-2 replacement. Amino group in AG contained two reactive hydrogens (H), and after one H reacted with ECH, the formed steric hindrance would lower the activity of another H. In addition, AG had long molecular chain, so it could be crimped more easily, and the possibility for amino N-2 replacement was lower. Therefore, amino N-1 replacement also took place under mass-to-charge ratios of 402 Da, 424 Da, and 440 Da, but the N-1 replacement was mainly amino N-1 replacement which occurred to two amino groups of AG molecules at the same time.

Fig.4 ESI-MS spectrum of the reaction products between ECH and AG

To further investigate structures of the ECH-AG reaction products and determine its carbon atom orders,DEPT-NMR (DEPT-90 and DEPT-135) was used to study the structures of ECH and AG reaction products as shown in Fig.5. Table 1 displays attributions of chemical structures of the main peak. As shown in Fig.5, AG had a long molecular chain, containing 8 carbons, where 1#, 5#, and 8# were carboxyl carbons,and 4# and 6# were CH. In addition, 1# and 4# were respectively connected to an amino group, amino group at 1# site could be easily exposed at the end position of molecular chain, and amino group at 4# site was located in the middle of AG. When long-chain AG molecules were crimped, their reactivity would be degraded.

Fig.5 13C-NMR spectra of the reaction products between ECH and AG

Table 1 13C-NMR analysis of the reaction products between ECH and AG

Under alkaline catalyzation, ECH firslty experienced ring-opening reaction, and amino groups of AG attacked carbon atoms at first as a nucleophilic reagent so that corresponding secondary derivatives were generated at the open ring induced by C-O bond breakage of ECH. Nucleophilic effect of alkali was higher than that of AG, so ECH would mostly experience hydrolysis reaction to generate chloropropanol under alkaline conditions.Chloropropanol obviously appeared in NMR spectrum.Chemical shifts 72.60 ppm, 64.01 ppm, and 47.97 ppm were CH and CH2.

In DEPT-135 spectrogram, peaks of CH and CH3were upward, while that of CH2was a negative peak.In DEPT-90 spectrogram, only upward CH peak could be seen. Chemical shifts 70.79, 70.16, 62.42, 58.62,and 55.86 ppm appeared simultaneusly in DEPT-90 and DEPT-135, indicating peaks of CH at the positions,where 55.86 ppm was CH absorption peak at 4# site of unreacted AG, 70.79 ppm and 70.16 ppm were CH absorption peaks from ECH open ring in the reaction product between AG and ECH, and 62.42 and 58.62 ppm were CH absorption peaks from AG in the reaction product between AG and ECH. 62.42 and 58.62 ppm had basically same integrals, manifesting that amino groups of AG reacted with ECH under the test conditions, but carboxyl or hydroxyl did not obviously react with ECH.

3.5 Discussion on modification mechanism of Camellia oleifera protein adhesive with EPR

The FT-IR spectra ofCamellia oleifera Abel.protein adhesive are shown in Fig.6. The absorption peaks ofCamellia oleifera Abel.protein in FTIR spectrum are mainly related to peptide bonds.C=O stretching vibration of amide-I band region at 1 656.5 cm-1, 1 515.8 cm-1came from bending vibration of N-H and stretching vibration of C-N of amide-Ⅱband region, and peak at 1 253.0 cm-1was C-N stretching vibration of amide-Ⅲ band region.-COOH at 1 375.1 cm-1was also observed. Comparing this withCamellia oleifera Abel. protein crosslinking with EPR, the decrease of peak at 1 375.1 cm-1and appearance of a new peak of ester carbonyl bond at 1 738.8 cm-1indicated that the carboxyl group of protein reacted with EPR. Red and blue shifts were found at absorption peak of amide-Ⅱ and amide-Ⅲ,moving to 1 550.4 and 1 248.6 cm-1, respectively,which indicated that the amino group ofCamelliaoleiferaprotein reacted with EPR to form a denser structure, and more energy was needed to complete vibration or expansion showed in FT-IR spectrum.

Fig.6 FT-IR spectra: (a) Camellia oleifera Abel. protein; (b)Camellia oleifera Abel. protein crosslinked with EPR

Based on the research results in this paper and a lot of early-stage research work regarding protein adhesives, the reaction mechanism of EPR-modifiedCamellia oleifera Abel.protein-based adhesive could be divided into two phases, namely resinification phase and curing phase as shown in Fig.7.

Fig.7 Reaction mechanism of Camellia oleifera protein adhesive by epoxy resin

In the resinification phase, the adhesive system was under solution environment, where chemical reaction was of selectivity, and EPR mainly reacted with amino ofCamellia oleifera Abel.protein. In the hot-pressing plywood preparation process (curing phase), moisture was rapidly volatilized, and the reaction of EPR had no selectivity, it mainly went through nearby reaction, so all active groups including-NH2, -COOH, OH, and so on, might react with EPR.

4 Conclusions

The development of a new protein-based adhesive withCamellia oleifera Abel.protein will be of great significance to improving the market competitiveness of protein-based wood adhesives. EPR-modifiedCamellia oleifera Abel.protein was used in this paper to prepare wood adhesive, and its performance and preparation mechanism were analyzed and discussed,and the results show that:

a) EPR could remarkably improve the bonding performance ofCamellia oleifera Abel. proteinbased adhesive. As the amount of EPR increased,the bonding strength presented firstly rising and then declining trend. When the amount was 9%, the bonding strength reached 0.72 MPa, which satisfied the strength requirement of Type II plywood in GB/T 17657-2013.

b)Camellia oleifera Abel.protein after alkali treatment could experience crosslinking reaction more easily with EPR with low curing temperature, high crystallinity of curing products and high degree of crosslinking reaction.

c) The reaction mechanism ofCamellia oleifera Abel.protein-based adhesive crosslinked with EPR could be divided into resinification phase and curing phase, where the former was of selectivity, mainly being amino reaction withCamellia oleifera Abel.protein, and the latter was of randomness, mainly being nearby reaction.

Acknowledgements

The authors highly appreciate the support program from the National Natural Science Foundation of China (Nos.31800481 and 31870546). The authors also thank the anonymous reviewers for their invaluable comments and suggestions to improve the quality of the paper.