Abstract
Non-rubber components are critical in the formation of the natural rubber (NR) vulcanization network, which leads to outstanding mechanical properties of NR. This study reports the effect of NR endogenous proteins (C-serum protein/lutoid protein [CSP/LP]) on the formation of vulcanization networks at the molecular level. Results indicate that CSP/LP has a positive effect on vulcanization. Fourier-transform infrared spectroscopy and X-ray photoelectron spectroscopy analyses demonstrate that the decrease in vulcanization time of CSP/LP is ascribed to coordination interaction between Zn2+ and amide bond. The interaction increases the availability of ZnO in the matrix, thereby promoting the formation of the vulcanized network. CSP/LP also participates in the construction of the vulcanization network as a new crosslinking point, thus increasing crosslinking density and improving the mechanical properties of the NR. This study provides new research ideas for studying the relationship among component–structure–property of NR materials and developing high-strength and high-toughness elastomer materials.
1 Introduction
The unique structure of natural rubber (NR) is the key to its excellent properties (1,2). NR has approximately 94% cis-1,4-polyisoprene and around 6% non-rubber components (NRC), like proteins and phospholipids (3,4,5). Although synthetic polyisoprene has a similar molecular chain structure to NR, its properties are inferior to NR (6,7,8). It shows that the molecular chain structure is not the only reason for the excellent comprehensive properties of NR, but the NRC also plays an important role. Thus, elucidating the role of NRC in the mechanical properties of NR is critical for comprehending the relationship between its structure, component, and properties.
Many studies indicate that the effect of NRC on NR properties cannot be ignored, since the mechanical properties of NR obviously decrease after the removal of NRC (9,10,18). In general, it has been proposed that the network structure formed by NR molecular chains increases the permanent entanglement points of the chains and promotes the strain-induced crystallization of NR, thereby improving its mechanical properties (10,11,12,13,14). After the removal of NRC (such as proteins and phospholipids), there are fewer permanent entanglement points, which reduces strain-induced crystallization degree (15). Many studies focus on the network structure affecting the green strength of unvulcanized NR, while the excellent comprehensive properties of NR mainly depend on the vulcanized crosslinking network structure (16,17). Thus, focusing exclusively on the influence of protein on the network structure of unvulcanized NR is insufficient to fully explain why NR has such superior mechanical capabilities.
At present, the research on proteins in NRC mainly focuses on the effect of exogenous proteins on properties. Studies have found that adding soy protein into purified NR limits the frictional behavior of the molecular chain, decreasing heat generation and improving the mechanical properties of NR (18,19,20). In addition, the improved interaction between soy protein and NR improves the mechanical properties after vulcanization (21). Nevertheless, considering the structural differences between exogenous and endogenous proteins, previous studies cannot fully elucidate the effect of proteins in NR on their properties. In this work, proteins (C-serum protein/lutoid protein [CSP/LP]) extracted from natural latex are quantitatively added back to centrifugal purification of natural rubber (CNR). Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) are utilized to characterize the coordination interaction between CSP/LP and Zn2+ and to evaluate the effect of protein on the formation of NR vulcanized crosslinking network at the molecular level. This work explores the contribution of NR endogenous proteins to its mechanical properties, and it is beneficial for a deep understanding of the relationship between NR components, structure, and properties.
2 Materials and methods
2.1 Materials
NR latex was obtained from Jinshui Processing Branch, Hainan Natural Rubber Industry Group Co., Ltd; tris-saturated phenol, Rainbow 180 broad-spectrum protein marker, 30% gel making solution, etc. from Beijing Solarbio Technology Co., Ltd.; ammonium persulfate, sodium dodecyl sulfate (SDS), sulfur, 2-mercaptobenzothiazole (accelerator M), stearic acid, zinc oxide, etc. from Aladdin Reagent Co., Ltd.; and hydrochloric acid from Xilong Chemical Co., Ltd.
2.2 Sample preparation
The NR endogenous proteins CSP/LP were prepared according to the published research method (22). The fresh latex was diluted to 30%, 0.5% SDS was added, and centrifuged at 20,000 rpm for 60 min at 4°C after that. The upper layer of cream was repeated to obtain secondary centrifuged natural latex and diluted to 30% after that. Raw rubber was produced by adding different amounts (0.5%, 1.0%, 1.5%, and 2.0%) of CSP/LP to secondary centrifuged natural latex and vacuum drying. The blend was prepared by mixing in two rolls in an open mill, and then, the vulcanized NR was produced according to the optimum curing time t 90. The curing formula is as follows: sample, 100 phr; stearic acid, 0.5 phr; accelerator M, 0.7 phr; and sulfur, 3 phr.
2.3 Characterization
2.3.1 Characterization of protein content and molecular weight
The concentration of extracted proteins was determined by the Bradford method using Tecan microplate (Infinite M200 PRO NanoQuant). The protein content of raw rubber was measured using a K9860 automatic Kjeldahl nitrogen tester; 30 μL of protein solution was mixed with 5 μL of 5 × electrophoresis loading buffer and denatured at 100°C for 5 min, and then, the protein mixture was separated in a 10% separation gel at 60 and 120 V.
2.3.2 Determination of vulcanization characteristics
The curing characteristics of the samples were evaluated in the rotorless vulcanizer (M-3000U, GOTECH). Test parameters are set as follows: rotation angle, 0.5°; frequency, 1.67 Hz; test time, 60 min; and test temperature, 145°C.
The vulcanization curves of the samples were measured by a rotorless vulcanizer at temperatures of 135°C, 145°C, 155°C, and 165°C. With the Arrhenius equation (Eqs. 1–3), activation energy E a of vulcanization induction period of NR samples with different amounts of CSP/LP can be obtained as follows:
where M H is the maximum torque value for curing, M t is the torque value corresponding to a curing time of t, t 0 is the time t corresponding to the minimum torque value for curing, A is a constant, k is the reaction constant, and α is the correction factor.
2.3.3 Coordination bond characterization
FTIR (TENSOR27, PerkinElmer, USA) and XPS (Axis Supra, Shimadzu, Japan) were used to analyze the coordination bonds formed between CSP/LP and Zn2+. Analysis by FTIR: the scanning wavenumber range is 4,000–400 cm−1, and the total number of scans is 16; analysis by XPS: C1s and N1s spectra are evaluated, and the binding energy of C1s for calibration is 284.8 eV.
2.3.4 Crosslinking density measurements
Crosslink density was determined with the equilibrium swelling method. The sample (accurate weight, m 1) was taken in 100 mL of toluene. The solvent on the surface of the samples was removed by filter paper after 7 days of swelling for accurate weighting (m 2). Calculate the crosslink density of vulcanization NR using Flory–Rehner equation (18).
2.3.5 Mechanical property measurements
Mechanical properties were measured using an AI-3000 tensile testing machine. Samples were cut into dumbbell-shaped specimens with a stretching speed of 500 mm·min−1. Dynamic mechanical properties of the vulcanization NR were measured by a dynamic mechanical analyzer (DMA) from TA Instruments in the United States. Strain amplitude: 15 μm; preset force value: 0.01 N; frequency: 1 Hz; heating rate: 3°C·min−1; and weep temperature: from −100°C to 100°C.
3 Results and discussion
3.1 Extraction and characterization of CSP/LP
Obtaining high-quality NR latex proteins proved to be difficult due to the viscosity and complexity of NR latex. In order to investigate the NR endogenous protein's contribution to the formation of its vulcanization network, it is necessary to extract the protein from NR latex first. As seen in Figure 1, 8,787 μg·mL−1 of C-serum protein and 5,749 μg·mL−1 of lutoid protein are extracted, respectively. Among them, the content of protein obtained from purified C-serum is more than the protein extracted by Wang et al., and about ten times more than the protein extracted by Li et al. (22,23). The C-serum protein and lutoid protein samples obtained in this study are highly purified, and it can be added to CNR as endogenous proteins to investigate their effect on the formation of NR vulcanization crosslinking networks.
(a) Three fractions of NR latex are centrifuged; (b) the protein content of C-serum and lutoid.
3.2 The effect of CSP/LP on vulcanization process
We added different contents of CSP/LP to CNR to investigate the effect of CSP/LP on vulcanization characteristics and vulcanization crosslinking network structures of NR. The t 90 of CNR decreases from 21.73 to 10.35 min after adding 2.0% CSP/LP, and the t 10 decreases from 4.12 to 1.33 min (Figure 2). With the increasing concentration of CSP/LP in NR, we significantly reduce the vulcanization induction period and maximum positive vulcanization time. The speed of the vulcanization increased significantly. Thus, CSP/LP has a positive effect on the vulcanization of NR. Meanwhile, after adding 2.0% CSP/LP, the maximum torque value of CNR increases from 2.48 to 4.36 dN·m. The addition of CSP/LP improves the vulcanization degree of NR, which in turn increases its torque value.
(a) NR protein content with CSP/LP addition; (b) vulcanization characteristic time of NR with different CSP/LP contents; (c) NR torque values of CSP/LP; (d) vulcanization curves of NR with different contents of CSP/LP; (e) E a of CNR and y% CSP/LP during the vulcanization induction period (y is the proportion of CSP/LP).
The effect of CSP/LP on the vulcanization of NR is primarily reflected in the induction period for vulcanization (Figure 2). In this article, the vulcanization curves of NR samples at different temperatures are measured using a rotorless vulcanizer, E a of the vulcanization induction period is calculated using the Arrhenius formula, and the impact of CSP/LP on the vulcanization induction period of NR is examined. By adding 2.0% CSP/LP, the induction period E a decreases from 86.14 to 81.21 kJ·mol−1 (Figure 2e). The results indicate that CSP/LP reduces E a of NR during the vulcanization induction period, accelerating the reaction rate of the vulcanization induction period. This result indicates that the NR endogenous protein CSP/LP has positive effects on the vulcanization process of NR.
3.3 The formation of coordination bonds
The NR vulcanization induction stage is mainly the result of the reaction between the vulcanization accelerator, the active agent, the sulfur, and the formation of Bt–S–Sx–Bt polysulfide bonds (24). The polysulfide bond structure reacts with the unsaturated double bond of the NR molecule chain to form a crosslinking precursor. In the vulcanization induction process, ZnO in the vulcanization accelerator provides soluble Zn2+ to chelate with the crosslinking precursor, which protects the weak bonds and generates short crosslinking bonds to increase the reaction site of the crosslinking process (25). The CSP/LP protein molecular chain has a significant number of peptide bonds. The Zn2+ combined with the amide group of peptides forms a more stable ligand, thereby increasing the utilization of ZnO within the matrix. The ligand enhances the active sites of the crosslinking reaction, promotes the crosslinking process, makes vulcanization more efficient, and encourages the vulcanization reaction (Figure 3a).
(a) The mechanism of promoting sulfidation by adding zinc oxide and CSP/LP; (b) FTIR and XPS of CSP/LP and ZnO/accelerator M/protein complex.
To verify the coordination interaction between CSP/LP and Zn2+, FTIR and XPS are used to analyze the interaction between CSP/LP and Zn2+. Following mixing CSP/LP, ZnO, and accelerator M at 145°C for 11 min, the characteristic absorption peak of the amide I band of the CSP/LP at 1,657 cm−1 shifts to the lower wavenumber of 1,638 cm−1, and the peak shape changes (Figure 3b). The result demonstrates that the oxygen and nitrogen atoms of the amide group of CSP/LP are used as power-supply groups to form coordination with Zn2+, resulting in a redshift of the characteristic FTIR peak. In coordination compounds, the element-binding energy depends on the state of oxidation of the element and the transfer of electrons from ligands (24). Hence, the binding energy of atoms is used to characterize metal ions and ligands in this study. The binding energy of N1s decreases from 400.1 to 399.4 eV after the CSP/LP, ZnO and accelerator M mixed thermocompression reaction. Since the metal–ligand anti-π bond increases the density of the electron cloud that coordinated in the ligand, and the binding energy is reduced (26). Thus, the decrease in N1s binding energy further indicates that the amide group of CSP/LP forms a coordinate bond with Zn2+. In conclusion, the amide bond of CSP/LP forms a coordination interaction with Zn2+ in ZnO, which improves the utilization rate of ZnO in NR vulcanization crosslinking system and promotes the formation of a vulcanized network.
3.4 The construction of the vulcanization network
The equilibrium swelling method is utilized to determine the swelling ratio and average molecular weight between crosslinking points of vulcanized NR with different CSP/LP contents. It also characterizes the degree of the vulcanized crosslinking network. The M c decreases from 19,237 to 11,255 g·mol−1 after adding 2% CSP/LP, indicating that the crosslinking density increased (Figure 4), since CSP/LP reduces the activation energy of NR vulcanization induction time and accelerates the vulcanization reaction. The number of crosslinking points formed by NR and sulfur increase, and the degree of NR crosslinking network is improved, which is also manifested in the increasing crosslinking density.
(a) Swelling ratio of vulcanized NR with different protein contents; (b) M c of vulcanized NR with different protein contents.
The NR endogenous protein CSP/LP has a large number of characteristic groups such as carboxyl (–COOH) and hydroxyl (–OH) that can be converted into thioester (–CSOR), thiol (–SH), and dithiocarboxylic acid (–CSSR) groups through sulfur reaction (27). The –CSOR, –SH, and –CSSR groups continue to react with accelerators to form polysulfide intermediates, which then react with different NR molecular chains to form vulcanization crosslinked networks (Figure 5). CSP/LP participates in the vulcanization process as a new point of crosslinking to participate in the formation of the vulcanized crosslinking network. Therefore, CSP/LP increases the number of crosslinking points within the vulcanized crosslinking structure and the crosslinking density of NR is increased.
Schematic diagram of protein accelerating vulcanization process.
3.5 The effect of mechanical properties induced by CSP/LP
The tensile strength of the vulcanization NR increases from 6.72 to 14.97 MPa after the addition of 2.0% CSP/LP (Figure 6a and b). The results show that CSP/LP increases the number of crosslinking points in the vulcanized crosslinking network structure, resulting in increasing crosslinking density and improving mechanical properties of vulcanized NR. With the increase of CSP/LP content, the storage modulus E′ of vulcanized NR in the glassy and rubbery regions increased significantly, and the glass transition temperature T g of the sample increases from −50.10°C to −45.72°C (Figure 6c and d). The results indicate that the vulcanization crosslinking point increases with CSP/LP addition increases, which leads to the restricted molecular chain movement and increases the T g of vulcanized NR. This study also confirms that CSP/LP changes the vulcanization crosslinking network structure by participating in the vulcanization reaction and then regulates the mechanical properties of NR.
(a) Stress–strain curves of vulcanized NR with different protein contents; (b) tensile strength of vulcanized NR with different protein contents; (c) the temperature-dependent curves of tan δ for samples with different protein contents; (d) the temperature-dependent curves of Eʹ for samples with different protein contents.
4 Conclusions
To conclude, we successfully extracted CSP/LP from NR and used them to study the mechanism of NR endogenous protein in accelerating NR vulcanization networks. Results show that the CSP/LP reduces E a of vulcanization induction and accelerates the vulcanization reaction. The accelerated vulcanization occurs because of the coordination interaction between amide bonds and Zn2+, which increases the utilization rate of ZnO in the matrix and promotes the formation of vulcanization networks. In addition, The CSP/LP acts as a new crosslinking point for the vulcanized crosslinking network, increasing the crosslinking density and improving the mechanical properties of NR. This work provides new research ideas for investigating the relationship between NR components, structures, and properties, also for developing high-strength and high-toughness elastomer materials.
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Funding information: This research was funded by the Strategic Priority Research Program of the Chinese Academy of Sciences (no. XDC06010100), Hainan Province Postgraduate Innovation Research Project (no. Hys2020-31), and Hainan Provincial Natural Science Foundation of China (no. 521RC1038).
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Author contributions: Xiu-Xiu Liu: writing – original draft, investigation, writing – review and editing, methodology, experiment, data analysis and plotting; Meng-Fan He, Ming-Chao Luo, Yan-Chan Wei: methodology, data analysis, formal analysis; Shuangquan Liao: writing – review and editing, resources.
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Conflict of interest: Authors state there is no conflict of interest.
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