A rheological study on non-rubber component networks in natural rubber

Abstract

Phospholipids and proteins were separately removed as the main non-rubber components to individually study their effect on the structure and properties of the rubber. Fourier Transform Infrared Spectroscopy (FTIR) and ¹H nuclear magnetic resonance spectroscopy (¹H-NMR) were used to characterize the chemical structure and the residual non-rubber component. A rheology study and stress relaxation measurements were used to study the role that non-rubber components play in natural networks. A rheological study showed that natural rubber (NR) and deproteinized natural rubber (DPNR) exhibited similar dynamic modulus at 170 °C. The lack of superposition in van Gurp–Palmen (vGP) curves at different temperatures for NR and DPNR, together with the shape of vGP curves, proved that long chain branching was mainly constructed by phospholipids. Stress relaxation measurements at room temperature were fitted with the Maxwell model and showed that the NR relaxation curve underwent a quick decrease and then came to an equilibrium stress retention of 58%, which is about 3 times higher than that of DPNR, indicating that proteins in NR contributed to the network structure at room temperature. Combining molecular dynamic studies, the interaction of proteins and phospholipids in non-rubber networks was proposed.

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Introduction

Natural rubber (NR) has been a particularly interesting and attractive material since it was first vulcanized by C. Goodyear in 1839. Natural rubber (NR) latex, collected from Hevea trees as a colloidal suspension,¹ is the main commercial feedstock for the rubber and latex dipping industries. The tire industry is one for which the applications of natural rubber are highly attractive, and obvious superiorities in mechanical and universal properties in a wide temperature range could allow such materials to be employed as innovative solutions to satisfy the demanding requirements that a modern-day tire must fulfill, such as wet grip, mechanical and thermal resistance, low cost, recyclability, etc.^2,3 Nowadays, natural rubber has become increasingly important for applications spanning many fields of chemistry, the hygiene and medical sectors, and it is further involved in a broad spectrum of consumer products in our daily life.^4–7

As a natural material, the yield of natural rubber which is seriously restricted by land resources and natural conditions, has become the fatal bottleneck for its extensive application. Thus, the synthetic analogue polyisoprene as the best alternative, is modifed mainly by various molecular design methods,^8–13 however the properties were disappointing.

It has been reported that non-rubbers,¹⁴ especially proteins and lipids, give natural rubber latex excellent properties which are unsurpassed by any synthetic latex.¹⁵ As a consequence, great industrial and scientific attention has been paid to the structures of non-rubbers and their importance on the structure of natural rubber to create the magical mechanical performance,¹⁶ such as the high strength of raw rubber (green strength), shape retention, high tensile strength, high crack growth resistance and minimal heat build up in the vulcanized state. Several attempts have also been made to study non-rubber components and investigate their corresponding structures in natural rubber. Y. Tanaka^17,18 first proposed that cis-polyisoprenes obtained from H. brasiliensis and Parthenium argentatum consist of more than 5000 isoprene units and that there are trans-isoprene units per rubber chain. The mainstream view now is that NR molecules comprise 2 trans-isoprene units connected to a long chain of cis-isoprene units. Two terminal groups, referred to as α and ω, have been postulated to link with mono- and di-phosphate groups associated with the phospholipids by H-bonding at the α-terminal, whereas the ω-terminal is postulated to be a modified dimethylallyl group linked to the proteins by H-bonding.¹⁹ Then the non-rubbers can connect the linear polyisoprene chains in NR through functional terminals and generate a branching or network topology, which is designated a “naturally occurring network”.²⁰ But since the Goodyear medal¹⁹ in 2001, no clear evidence of the structure of an end-linking network has been reported.

Although the real cross-linking structure and role of non-rubbers are not fully understood, it has been suggested that the cross-linking of NR formed by non-rubbers can be eliminated by deproteinization and transesterification. After the removal of proteins and phospholipids, linear rubber chains can be obtained.²¹

The effect of proteins and phospholipids on strain induced crystallization and the correlation with the tensile strength of NR have been extensively studied using X-ray diffraction and microscopic methods.^22,23 However, spectroscopy methods are static and mostly accompanied by solvent, and X-ray characterization is indirect in “naturally occurring” network characterization. Rheological properties are highly sensitive to long chain branching because even a low degree of long chain branching has a significant effect on the viscoelasticity in polyethylenes^23–28 or polypropylene.²⁹ A van Gurp–Palmen curve, which was first proposed to judge the feasibility of Time–Temperature Superposition (TTS) principles and then evolved to branched structure studies independent of the chemical components of the macromolecule chain, is also very sensitive to the long chain branching of the polyisoprene melt in unvulcanized NR.³⁰ Therefore, the rheological behavior of unvulcanized NR can be characterized using a dynamic spectrum, related to the molecular structure in the melt, which is strongly impacted on by branching or networking. However, to our knowledge, there is a lack of a systematic investigation on how non-rubber components affect the rheological behavior of rubber chains in melts and inversely testify the role of non-rubber components from rheology, especially the effect of long chain branching on rheological properties. While few rheological studies were conducted on NR, among which most focused on blended NR with plastic,³¹ silica,^32,33 carbon black³⁴ or another natural ingredient³⁵ in a vulcanized regime, hardly any were on unvulcanized rubber. While for unvulcanized NR, the studies mainly focused on processability³⁶ or nonlinear viscoelastic behavior.³⁷ In this study, we conducted rheological studies on an unvulcanized NR melt, and introduced the widely used vGP curve to study the effect of non-rubber components on unvulcanized NR.

In addition, the stress relaxation behavior also provides important guidance in probing the network, and additionally the equilibrium relaxation modulus G_∞ is proportional to the crosslinking density.²⁸ The stress relaxation behavior of natural rubber is often investigated for vulcanized rubber,^29–31 while scarcely for the unvulcanized if any. In this study, to investigate the network structure constructed by non-rubbers in unvulcanized NR, we introduced a stress relaxation test and analyzed the equilibrium relaxation modulus to evaluate the crosslinking density and the constraints on relaxation units.

In this work, an attempt was made to clarify the “naturally occurring network” through a molecular dynamics study, combining the chemical structure analysis. Fourier Transform Infrared Spectroscopy (FTIR), rheology and stress relaxation were used to analyze the composition and structure of the natural network. To individually study the effect of the non-rubber components, three models were prepared to individually study the effect of non-rubber components which are listed as follows, NR containing all of the non-rubber components, deproteined natural rubber (DPNR) containing phospholipids and fatty acids, transesterified deproteinized natural rubber (TEDPNR) containing only rubber chains.

Experimental

Materials

Natural rubber latex used in the present work was commercial high ammonia natural rubber (HANR) latex purchased from Dongfeng company (China). Total solid content and dry rubber content of the HANR latex, determined according to ASTM D 1076, were 61.3% and 60.8 w/w%, respectively. Triton X-100 and protease (P-5380) was provided by Sigma-Aldrich. Other reagents used were analytical grade.

Preparation of specimens

Three models were prepared to individually study the effect of the non-rubber components which are listed as follows, natural rubber containing all of the non-rubber components, deproteined natural rubber (DPNR) containing phospholipids and fatty acids, transesterified deproteinized natural rubber (TEDPNR) containing only rubber chains.

Natural rubber

HANR latex was centrifuged once for the removal of debris, and then spread into a thin layer to dry under reduced pressure at ambient temperature until it remained a constant weight in a vacuum oven.

Deproteinization of natural rubber

Deproteinization of the HANR latex was carried out through incubation with 0.08 wt% protease in the presence of 0.15% v/v Triton X-100 and 1 wt% aqua ammonia at 37 °C for 12 h. The dry rubber content was 30%. The cream fraction was redispersed in the surfactant, in which the dry rubber content was adjusted to 30 wt% with deionized water. It was washed two or three times by centrifugation. The rubber was recovered by centrifugation followed by coagulation with methanol and dried under reduced pressure at ambient temperature until it remained a constant weight.

Measurement of the nitrogen content of the deproteined natural rubber (DPNR) was made using a nitrogen elemental analyzer (CARLO ERBA 1106, Italian), and the nitrogen content was reduced from 0.5% to 0.07% after deproteinization.

Transesterification of the deproteined rubber

Transesterification of the rubber from DPNR was carried out in 2% w/v toluene through reaction with freshly prepared sodium methoxide and stirring at room temperature for 8 h. The resulting transesterified deproteinized natural rubber (TEDPNR) was purified by precipitation of the rubber solution using an excess of methanol and then dried in a vacuum oven.

Measurement of the phosphorus content of TEDPNR was made using a phosphorus elemental analyzer (IRIS 1000 ICP-AES, Thermo Electron Co. USA), and the phosphorus content was reduced from 224 ppm to 65 ppm after transesterification.

The molecular weight of all samples was determined using gel permeation chromatography (HLC-8320, Waters), and the number averaged molecular weight was about 150–160 kg mol⁻¹ for NR and DPNR, 134 kg mol⁻¹ for TEDPNR, while the MW of commercial linear isoprene rubber (IR) was 317 kg mol⁻¹.

Characterization

FTIR. The rubber samples for FTIR analysis were prepared by casting 1% w/v solutions of the rubber in tetrahydrofuran (THF) on a KBr disk, and analysis was conducted with a Nicolet-560 infrared spectrometer (USA) using a potassium bromide (KBr) pellet. FTIR spectra were recorded in the spectrumal range of 4000–400 cm⁻¹ with a 2 cm⁻¹ resolution and 32 scans.

¹H-NMR. ¹H-NMR spectra were recorded on a spectrometer (Varian INOVA-400) at 25 °C using CDCl₃ as the solvent and TMS as the external standard.

Rheology analysis. Dynamic mechanical measurements were performed using a HAAKE PXR800 rheometer. Shear deformation was applied under the conditions of controlled deformation amplitude, always remaining in the range of linear viscoelastic response. Frequency dependencies of the storage (G′) and loss (G′′) shear moduli were measured in the frequency range 0.01–100 Hz at a deformation amplitude ranging from 2 to 4% depending on temperature and at various temperatures between 160 °C and 190 °C. The geometry of parallel plates was employed with plate diameters of 20 mm and a sample thickness of 1.2 mm. Temperature control (0.1 °C) was achieved via a water circulation system. Before measurements, the samples were processed into even thickness plates through heat-pressing at 150 °C for 5 min in a mold and then isothermally for 12 h under vacuum at 40 °C.

Stress relaxation. Stress relaxation was conducted in a INSTRONG universal tensile test machine at a strain of 20%. The total length was 15 mm and elongated to the target strain at a 500 mm min⁻¹ tension rate and then the strain was held constant for 1200 s. Before measurements, the samples were processed into even thickness plates through heat-pressing at 150 °C for 5 min in a mold and then isothermally for 12 h under vacuum at 40 °C.

Results and discussion

Chemical structure characterization of the NR samples

Infrared spectroscopy was used as a method for chemical structure analysis, and also to study intermolecular interactions caused by hydrogen bonding, because the vibrational modes of the donor and acceptor groups are sensitive to this interaction leading to a change in the vibrational characteristics.³² The FTIR spectra of NR, DPNR and TEDPNR at room temperature are plotted in Fig. 1.

Fig. 1 FTIR spectra of NR, DPNR and TEDPNR.

First, a wide H-bond peak at around 3400 cm⁻¹ was absent in TEDPNR. For NR the wide peak comprised a relatively sharp peak at about 3300 cm⁻¹ related to the N–H symmetric stretch of the H-bond, and the wide H-bond peak is mainly ascribed to the O–H symmetric stretch in the H-bond in non-rubbers, overlapping with a small amount of environmental water absorbed by non-rubbers.

Second, to pick out the non-rubbers, the assignment of the rubber chain peaks are listed below. v_max/cm⁻¹ = 836 (trisubstituted olefin out-of-plane =CH, wag), 1129 (–CH₃, rock), 1300 (–CH₂–, wag), 1376 (–CH₃, symmetric deformation), 1450 (–CH₂– symmetric and –CH₃ asymmetric deformation), 1664 (C=C, stretch), 2720 (overtone of –CH₂– umbrella), 2850 (–CH₂– and –CH₃, symmetric stretch), 2920 (–CH₂–, asymmetric stretch), 2962 (–CH₃, asymmetric stretch), and 3030 (olefin =CH– stretch).^33,34

The recession of the characteristic protein peaks at 1663 and 1546 cm⁻¹ (ref. 35) is observed in DPNR compared with NR, and the C=O phospholipid peaks at 1738 cm⁻¹ (ref. 36) in TEDPNR. The FTIR results, in combination with the elemental analysis in Section 2, proved that the intended samples were successfully prepared. As for the different intensity of the C=C stretch peak in the normalized infrared spectra, it can be interpreted as occurring due to various infrared activities with different non-rubber environments.

The shoulder peak at 1260 cm⁻¹ in NR was shifted to 1248 cm⁻¹ and became sharp after protein removal, continually shifting to about 1243 cm⁻¹ and receding rapidly after phospholipid deprivation. It has been reported that the asymmetric O–P–O vibrational band is extremely sensitive to hydration, and the O–P–O asymmetric stretching of the hydrated phospholipid bilayer is usually observed at 1230 cm⁻¹, whereas dried or anhydrous lipid always appears at a 30 cm⁻¹ higher frequency.³⁷ The shift of the O–P–O asymmetric stretching peak strongly suggests that the phospholipids in NR interacted with the protein via H-bonds, and in DPNR rubber they aggregate or link together via H-bonds.³⁸

¹H-NMR

The ¹H-NMR spectra of NR, DPNR and TEDPNR are shown in Fig. 2, and a small residual chloroform peak was detected at 7.28 ppm. All of the spectra were normalized using the =C–H at the backbone triplet (J = 6.5 Hz) peak at 5.15 ppm. A small multiplet signal resonating at 3.91 ppm was expected to be derived from the nonequivalent methylene protons linked to a phosphate group, –CH̲₂OP–.³⁹ The multiple peaks at 2.365 ppm, 2.375 ppm and 2.395 ppm were assignable to the –CH₂– of long-chain fats, the multiple peak at 2.06 ppm was assignable to –CH̲₂– in the backbone, the single peak at 1.7 ppm was assignable to –CH̲₃ in the backbone, the multiple peak at 1.43 ppm was assigned to the –CH̲₂– of protein, and the multiple peak at 1.13 ppm was assigned to the –CH̲₃ of protein. From the extended spectra we can see that protein was removed after deproteinization, long-chain fat was eliminated after transesterification, while phospholipid was partly removed in TEDPNR. This can be interpreted by the suggestion that some phospholipid was connected with the rubber chain terminal.¹⁶

Fig. 2 (a) ¹H-NMR spectra of NR, DPNR and TEDPNR in the range of 0–7.5 ppm, extended ¹H-NMR spectra of NR, DPNR and TEDPNR normalized using the 5.15 ppm peak in the range of (b) 4.9–5.4 ppm, (c) 1.10–1.16 ppm, (d) 1.39–1.47 ppm, (e) 3.80–4.00 ppm, and (f) 2.34–2.42 ppm.

Combining the ¹H-NMR and FTIR results, we can conclude that the intended samples were successfully prepared.

Branched molecular structure in unvulcanizated natural rubber

To further study the effect of non-rubbers on the main chain structure, the rheological behavior is analyzed in this Section.

All measurements were conducted at a fixed strain, ranging from 2% to 4% at 170 °C where all samples display a linear viscoelastic performance, and the storage modulus G′ as a function of frequency is shown in Fig. 3. At such a high temperature, most H-bonding or other non-bonding interactions were destroyed at this temperature.

Fig. 3 Dynamic spectra of NR, DPNR and TEDPNR at 170 °C.

NR and DPNR show a higher modulus at low frequency than TEDPNR, indicating the existence of long-chain branching, crosslinking or phase-separation.^40,41 However, the modulus of NR is a little higher than that of DPNR. DPNR displayed no apparent slope change at low frequency, while the modulus increased by as much as 2 orders of magnitude in contrast with TEDPNR.

To further investigate the modulus change, dynamic spectra at different temperatures were obtained for all of the samples, and van Gurp–Palmen plots were drawn using dynamic shear modulus values at three different temperatures (160, 170, and 190 °C), displayed in Fig. 4. Generally, very good superposition of the vGP curves was observed for classical viscoelastic polymers, while a small amount of long chains can significantly distort the vGP curve and thus cause a misalignment.^42,43 The rubber models were confirmed to be thermo-rheological melts from the discrepancy of the vGP curves for NR and DPNR in Fig. 4, possibly with long-chain branching or phase-separation.

Fig. 4 vGP curves for (a) NR, (b) DPNR and (c) TEDPNR at various temperatures.

To judge the reason for the discrepancy in the vGP curves, a temperature sweep from 100 °C to 200 °C at a frequency of 1 Hz at 1% strain was conducted. As is known, phase-separation can cause a sharp uprush in the modulus of the temperature sweep.^44,45 But here, the results show no uprush in the dynamic modulus in Fig. 5, indicating that no phase separation occurred in the testing temperature region. As is shown in Section 2, all samples were unvulcanized, so we can exclude crosslinking. After excluding phase-separation, the modulus shift of NR and DPNR in Fig. 3 is attributed to long chain branching, and a small amount of protein involved in forming long-chain branches.

Fig. 5 Temperature sweep at a frequency of 1 Hz at 1% strain from 100 °C to 200 °C.

In contrast to NR and DPNR, the overlap of the vGP curves at the various temperatures is good for TEDPNR, indicating that TEDPNR is a simple fluid. From the shape of the curve – only one minimum, spread monotonically to 90° in the phase angle – we can be conclude that TEDPNR was probably linear. Combining the good linearity of the Han map⁴⁶ in Fig. 6, we can conclude that the curve for TEDPNR was linear at high temperature, in accordance with the dynamic spectrum.

Fig. 6 Han map of DPNR and TEDPNR at 170 °C.

The above rheological results showed that long-chain branching existed only in the NR and DPNR melt, indicating that phospholipids and a small amount of protein acted as the branching point in the long-chain branching construction. Furthermore, we can conclude that phospholipids and a small amount of protein connected with the rubber terminal point by chemical bonds or very strong interactions (which survive at 170 °C), then the non-rubbers coagulate through micellar interaction,⁴⁷ and possibly formed the star structure in the melt.⁴⁸

Network structure in unvulcanizated natural rubber

Rheological experiments on the structure of the three models at high temperature confirms the stable topology in NR, which proves in turn that the natural network is further constructed mainly through thermally unstable H-bonding or non-bonding interactions between the non-rubber components and rubber chain ends, in accordance with the FTIR results. However, does the natural network exist in melts at room temperature? To answer this question, stress relaxation tests were conducted. As is known, stress relaxation analysis is an effective method for studying the network, and the equilibrium relaxation modulus G_∞ is proportional to the crosslinking density.⁴⁴

In this study, the stress relaxation curves of NR, DPNR and TEDPNR at 25 °C are displayed in Fig. 7, where all of the samples were quickly subjected to a strain of 20% to avoid the influence of strain induced crystallinity (SIC),⁴⁹ and then the strain was maintained for 1200 s. For the sake of contrast, a relaxation curve of fairly linear IR with a 317 kg mol⁻¹ average molecule weight (MW) was added. Generally, the stress of linear viscoelastic polymers quickly relaxed to a very small value, and even to zero.

Fig. 7 Stress relaxation curves of NR, DPNR, TEDPNR and IR at 25 °C.

To illustrate the effect of non-rubbers on the stress relaxation tendency, all of the instant stress values were normalized using the corresponding initial stress, as is displayed in Fig. 7. The relaxation curves of NR tended to form a plateau after about 300 s and the retention is more than 50%, the largest stress retention, indicating that protein mainly contributed to the natural network at room temperature. Unexpectedly, DPNR produced nearly the same behavior as IR, but relaxed slightly slower than linear IR. TEDPNR relaxed to zero during the testing period. It can be confirmed that the relaxation units in NR were obviously confined by a chemical or strong physical network. While in DPNR, in contrast to NR the network constraint was scarcely observed. However, in comparison with linear IR, we can conclude that the restriction derived from long chain branching. TEDPNR displayed typical relaxation behavior of linear molecular chains with fully relaxed stress.

To further confirm the above assumption, we adopted the Maxwell model^41,50,51 to describe the stress relaxation of rubber, as shown in eqn (1):

In eqn (1), σ(t) represents the stress in the relaxation, σ_e is the equilibrium stress, σ_i is the coefficient of the ith Maxwell model and τ_i is the relaxation time of the ith Maxwell model.

Elastomer relaxation followed a 7-element Maxwell model,⁵² as shown in eqn (2):

Divided by the initial stress σ₀ on both sides, eqn (2) may then be inverted to read:

Let

Then we fitted the experimental data from eqn (3) and the results are listed in Table 1.

Table 1 The Maxwell fit result of stress relaxation at 25 °C

Sample	A 0	A 1	T 1	A 2	t 2	A 3	t 3	R ^2
NR	0.58507	0.06667	9.87772	0.13743	57.85191	0.18798	364.7487	0.9876
DPNR	0.15905	0.09697	12.62921	0.20228	82.65005	0.51581	544.9699	0.99534
IR	0.06292	0.13534	17.76458	0.27497	96.31696	0.50153	561.9759	0.99674
TEDPNR	−0.09715	0.21816	16.88401	0.33616	115.2694	0.48798	740.3617	0.9829

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From eqn (4), we can see that A_i represent the contribution of t_i, and t_i corresponds to the different relaxation unit in the samples. The values of A₁, A₂ and A₃ increased in the same samples with increasing the magnitude of the relaxation times t₁, t₂ and t₃. The consistency of A_i and t_i proved that the larger relaxation unit contributed to larger stress relaxation.

Second, the crosslinking density was discussed through A₀. As is shown in Table 1, NR displays the largest A₀, quadruple that of DPNR, while the A₀ of DPNR is also higher than that in linear IR, and TEDPNR shows a meaningless negative A₀.

From eqn (4) we can see that A₀ represents the stress retention rate, which is proportional to the equilibrium relaxation modulus G_∞ and crosslinking density, regardless of being chemical or physical. Then we can conclude that NR possessed the largest cross-linking density, and the long chain branches in DPNR constructed a small amount of a strong entanglement network. From the crosslinking density value which is 3 times higher, we can also conclude that part of the network structure in NR is connected by interaction between proteins. Combined with the FTIR study, we can conclude that the interaction between proteins and phospholipids can also contribute to the natural network, in accordance with previous studies.^53,54

For the linear IR, the super-high MW contributed to a strong entanglement network, but was still weaker than in DPNR. While in TEDPNR, the linear topology and lower MW results in the absence of a strong entanglement network.

Then the relaxation time was also considered. The relaxation time of all of the samples is displayed in the histogram in Fig. 8.

Fig. 8 Maxwell relaxation time histogram of NR, DPNR, TEDPNR and IR.

As is shown in Fig. 8, the relaxation times decrease in this order: NR < DPNR < IR < TEDPNR. In vulcanized rubber, the relaxation time decreases with increasing crosslinking density, which can be interpreted as the crosslinking point restriction.³⁸ Similarly, in the unvulcanized rubber, the relaxation units were also constrained by the natural network, and then displayed a decrease in relaxation time. The results confirmed that the crosslinking density increased in the order: NR > DPNR > IR > TEDPNR, which agreed with the previous study of A₀.

From the above results, we proposed the non-rubber in the structures of NR and DPNR shown in Scheme 1. In NR, the protein is connected in a ω-terminal and then coagulated with each other through H-bonds, part of which can also interact with phospholipids through H-bonds. Moreover, in the α-terminal, mono- or di-phosphate is bonded with the rubber chain, and then connected by a micelle cluster. While in DPNR, the phospholipids or protein fragments after the protease treatment can also interact with the ω-terminals by polar–polar interactions or H-bonds, together with the α-terminals being connected to phospholipids to form the network, whose density is much lower than that in NR.

Scheme 1 Assumed network structure in (a) NR and (b) DPNR.

Conclusions

The long chain branching points formed by phosphates, and crosslinking points consisting of proteins and phospholipids are confirmed through the rheological and stress relaxation changes of the three models. The modulus decreased at low frequency, and the equilibrium stress retention decreased by 75% after deproteinization, suggesting that the network was broken through removing the protein. The van Gurp–Palmen curves showed a very good overlap and the stress relaxed to zero after transesterification, indicating the decomposition of long chain branching points by phosphatase. The FTIR study of NR and DPNR further reveals that the H-bonding between proteins and phosphatase connected with the terminal phosphatide mainly contributed to the physical crosslinking in NR. While in DPNR, some of the phospholipids and protein fragments after the protease treatment may also be connected with the ω-terminals by H-bonds to form the weak network, and H-bonds between phosphatase and α-terminal phosphatide participated together in the formation of long chain branching points.

Acknowledgements

We acknowledge funding from the national natural science foundation of China to carry out this work (contract grant number 51333003). Authors must particularly acknowledge GS Huang for her forceful guidance and generous assistance.

Notes and references

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