Comparative Study of Natural Rubber Latex Particles and Artificially Prepared Rubber Particles

$Comparative Study of Natural Rubber Latex Particles and Artificially Prepared Rubber Particles$

Comparative study of natural rubber latex particles and artificially prepared rubber particles

and Amir-Hashim M.Y ٭ A.R. Ruhida , ٭,#Tae Han Shen ,٭.Shamsul Bahri A.R

Rubber Research Institute of Malaysia (RRIM), Malaysian Rubber Board (MRB), P.O. Box 10150 Kuala Lumpur, Malaysia ٭ # School of Science and Technology, Universiti Malaysia Sabah, P.O. Box 2073 Sabah Malaysia Abstract The bulk of the rubber in NR latex exists in the form of particles dispersed in the latex emulsion. These naturally occurring particles are made-up of rubber core surrounded by a layer of non-rubber materials such as protein and lipids. In this study, the electron microscopy technique was used to differentiate between the natural rubber particles and the artificially formed rubber particles prepared from dried latex compound. The artificial rubber particles appeared to be morphologically similar to the particles of natural rubber but with different particle size distribution. More importantly, the artificially rubber particles were not surrounded by rubber particle membrane proteins normally associated with natural rubber particles. The study is part of a concerted effort to develop NR-based synthetic rubber substitute in anticipation of future energy crisis associated with the dwindling global petroleum supply, the main raw material for many synthetic rubber. Key words: NR, natural latex, proteins, artificial latex, TEM, immunogold labelling, membrane protein Introduction Artificial isoprene latex, derived from of artificially formed rubber particles pre-formed polymers, normally derived and identifying the proteins membrane by re-dispersing bulk synthetic normally associated with the rubber polyisoprene cement in an aqueous particles of NR latex. medium. It has been said that physical Materials and Methods characteristics of the latex films prepared Preparation of artificial latex from dried from artificial synthetic cis 1,4- latex polyisoprene is similar to that of films Rubber cement was prepared by prepared from natural rubber latex immersing cut dried latex materials in a (Blackley D. C. 1997). Nevertheless, it is solvent bath containing analar-grade not known of the similarity between organic solvent and left to swell. The particles of artificially formed rubber swollen rubber cement is subsequently particles to NR latex particles. Thus, it is emulsified to give a stable rubber/solvent the intention of the paper to deliberate, in emulsion. The emulsion composition particular, on the sizes and distribution obtained is subjected to solvent removal 2

process, subsequently giving dilute temperature. Finally, visualisation of the rubber latex. Thereafter, the dilute latex immunogold labelled-rubber particles is then concentrated up to 30% total was done, using a TEM at 80 kV. solids content (TSC). Results and Discussion. Determination of rubber particle Micrographs 1 and 2 illustrate the property and size distribution. naturally occurring rubber particles of Osmium tetroxide stained rubber latex fresh field NR latex and of highly samples were visualised under ammoniated (HA) NR latex, transmission electron microscope at 80 respectively. The rubber particles were kV. The presence, size distribution and generally spherically shaped bodies morphological properties of the rubber while some were pear-like shaped. The particles for each of the samples were largest particle for the fresh field latex then determined using an image analyser sample (1320 nm) were similar in size to attached to the electron microscope. the largest particle of HA latex Double-immunogold localisation for proteins concentrate (1328 nm) while the smallest Osmium tetratoxide stained rubber latex particle (35 nm) was much smaller than samples was incubated with the first the HA latex concentrate particle (80 primary antibody, rabbit IgG anti-Hev b nm). This is expected for concentrated 1. Next, the samples were washed and NR latex because concentrating by incubated with the first secondary centrifuging process should remove antibody, goat anti-rabbit IgG small and light particles. conjugated to 10 nm gold particles. In terms of their size distribution, rubber Then after another series of washings, particles in the size range of between incubation with primary antibody, rabbit 100 nm to 200 nm were the most IgG anti-Hev b 3, was done followed by abundant in fresh field latex (Figure 1) washings and incubation with the second and these particles made up 43.2 % of secondary antibody of goat anti-rabbit the total rubber particles observed. On IgG conjugated to 30 nm gold particles. the other hand, the range of rubber After another series of washings, each particles were between 200 nm to 300 sample grid was left to dry at room nm for concentrated HA latex (Figure 2). 3

It was worthy of note, the very large the artificial rubber particles were rubber particles of more than 1100 nm in created by inducing agglomerates from diameter were few in both latex. gel-like high solvent filled rubber and As for the artificial latex, the range of still the rubber shaped itself and particle sizes of the rubber particle distributed in the particle size range of obtained after removing most of its its natural counterpart. solvent content were more or less On the contrary, artificial rubber comparable to range of the naturally particles treated with soaps, namely 10 occurring NR rubber particles. The % rubber solvent emulsion (RSE) with 1 largest was 1574.59 nm while the part soap, 10 % RSE with 2 parts soap smallest was 42.86 nm. Rubber particles and 15 % RSE with 2 parts soap, were with diameters of between 100 nm to not morphologically similar to those 200 nm were the most abundant in the observed in the previous samples. In the sample comprising of 23 % of the total 10 % RSE with 1 part soap sample, its rubber particles, while, again those of the rubber particles tended to cluster, bigger particle sizes larger than 1300 nm forming a massive structure while still in diameter were the least observed maintaining their spherical shape (Micrographs 3 and 3a). property as depicted in Micrograph 4. In general, from these micrographs, Increasing the soap content of the 10 % despite the origin of these rubber RSE and 15 % RSE with 2 parts soap particles, particles with diameters content, the rubber particles were ranging from 100 nm to 400 nm, completely distorted. They were attached dominated the total rubber particles to each other, forming lumps and they counted. This finding correlated well were no longer globularly shaped as with the previously reported study by shown in Micrographs 5 and 6 Singh et al., 2003. The shape of the respectively. The collodion films of the rubber particles observed in this study two latter samples were covered with corresponding to those described by artifacts of what were believed to have Gomez and Hamzah, 1989. This come from the deformed rubber observation is quite interesting because particles. 4

The addition of soap as in the 10 % RSE small or the large rubber particles, with 1 part soap, 10 % RSE with 2 parts confirming the presence of this protein soap and 15 % RSE with 2 parts soap on the membrane of all of the rubber samples, had resulted in the clustering of particles regarding of their sizes. On the the rubber particles. By increasing the other hand, the 30 nm gold particle, presence of soap from 1 part to 2 parts, associated with protein Hev b 3, was the rubber particles were totally ruptured mainly detected on the small rubber and destroyed. Thus, suggesting that particles with diameters of below 400 soap used had an annihilate effect over nm. the rubber particles. Similarly, for the HA latex concentrate Double-immunogold localisation of sample, the 10 nm gold particles were proteins (Hev b 1 and Hev b 3) found to be present on both of the small Electron microscopy visualization and large rubber particles as shown in technique using different sizes of Micrograph 8. Again, the 30 nm gold electron opaque markers such as particles were seen to be on the colloidal gold had enabled the membrane of the small rubber particles. identification of different materials However, the amount of the 30 nm gold simultaneously in the same sample particles was significantly reduced as (Doerr-Schott and Lichtem, 1986; compared to those found in the fresh Robinson and Vandré, 1997; Takizawa field latex sample, implying that the et al., 1998; Yi et al., 2001). processing of concentration somewhat In the study, the marking of proteins on affects the protein nature of NR latex. NR rubber particles with electron- For the other two samples of artificially opaque gold particle enabled the prepared rubber particles, namely the visualisation of some of the protein distilled and creamed latex samples, no component associated with NR particle. labelling was observed as demonstrated The fresh field NR latex sample showed in Micrographs 9 and 10, respectively. the immunogold labelling for the The absence of these gold particles proteins (Micrograph 7). The small gold marked the diminishing of the proteins particles (10 nm), acted as the marker for associated with the rubber phase. The protein Hev b 1, were found on both, the 5

destruction of the proteins resulted in the technical assistances. The financial loss of antigenic sites for the gold- support for the project is provided by the conjugated antibodies. As the result, no Malaysian Rubber Board. labelling took place. References The rubber particles observed in the Blackeley D.C (1997), Polymer Latices, fresh field latex, HA latex concentrate, Science and Technology. Chapman and nd creamed latex and distilled latex samples Hall. 2 Edition . Vol 2., pp 394-437 were globular bodies and some were Doerr-Schott J. and Lichtem, C. M. (1986), Journal of Histochemistry and pear-like in shape. Small rubber particles Cytochemistry. 34 (8). 1101-1104. with diameters of less than 400 nm were Gomez, J. B. and Hamzah, S. (1989), the most abundantly found in these Journal of Natural Rubber Research. 4 samples. For the 10 % RSE with 1 part (3). 204-211. soap, 10 % RSE with 2 parts soap and 15 Robinson, J. M. and Vandré, D. D. % RSE with 2 parts soap, the rubber (1997), Journal of Histochemistry and Cytochemistry. 45 (5). 631-642. particles were totally distorted and Singh, A. P., Wi, S. G., Chung, G. C., clustered to each other. The Hev b 1 and Kim, Y. S., and Kang, H. (2003), Hev b 3 proteins were present on the Journal of Experimental Botany. 54 (384). 985-992. membrane of the rubber particles for the fresh field latex and HA latex Takizawa, T., Saito, T. and Robinson, J. M. (1998), Journal of Histochemistry concentrate samples. Hev b 1 was found and Cytochemistry. 46 (1). 11-17. on both the large and small rubber Yi, H., Leunissen, J. L. M., Shi, G. M., particles while Hev b 3 was localised Gutekunst, C. A. and Hersch, S. M. (2001), Journal of Histochemistry and mostly on the small rubber particles. Cytochemistry. 49 (3). 279-283. However, these proteins were not detected on any of the artificially formed rubber particles of the other samples. Acknowledgement The authors wish to thank Chan Jee Leene, Roslim Ramli, Juseph Ambrose and Zaharah Hanif for their excellent

Micrograph 1: Rubber particles of osmium tetroxide-fixed fresh field latex sample

Micrograph 2: Rubber particles of osmium tetroxide-fixed HA latex concentrate sample

Size Distribution of Rubber Particles in Fresh Field Latex

50.0 43.2 45.0 40.0 35.0 30.0

25.0 21.6 20.5 20.0 15.0 10.0 3.8 Relative Abundance (%) 2.3 2.3 5.0 1.8 1.8 0.9 0.5 0.7 0.2 0.2 0.2 0.0 1234567891011121314

Particle Size Class

Figure 1: Size distribution of rubber particles in fresh field latex

Size Distribution of Rubber Particles in HA Latex Concentrate

40.0 35.3 35.0 32.2 30.0

25.0 20.0

15.0

10.0 7.6 6.2 5.5

Relative Abundance (%) 4.8 2.8 5.0 1.4 1.4 1.4 0.7 0.3 0.0 0.3 0.0 1234567891011121314 Particle Size Class

Figure 2: Size distribution of rubber particles in HA latex concentrate sample 8

Migrographs 3a & 3b: Artificially formed rubber particles of osmium tetroxide-fixed a) after solvent removal and b) after concentration process.

14 .0 0 12.8 1 12 .8 1 11.7 4 12 .0 0 10 .6 8 10.3 2 9.96 10 .0 0

7.83 8.00 6.41 6.05 6.00

4.00 2.85 2.85 2.49 2.00 0.71 0.71 0.36 0.36 0.36 0.36 0.36 0.00 0.00 0.00 0.00 0.00 1234567891011121314151617181920212223 Particle Size Class

Figure 3: Size distribution of rubber particles of osmium tetroxide-fixed after solvent removal

Size Distribution of Rubber Particles in Distilled Latex

25.00 23.03

20.00 18.61 ) % (

e c n 15.00 11.67

bunda a

e 10.00 v 7.57 i t

a 6.31

e 5.05 5.36 5.36 R 5.00 3.79 3.79 3.79 2.21 1.26 0.95 0.63 0.63 0.00 12345678910111213141516 Particle Size Class

Figure 4: Size distribution of rubber particles of osmium tetroxide-fixed after the concentration process

Micrograph 4: Rubber particles of 10 % RSE with 1 part soap. Note the spherical shape of the rubber particles even though they were clustered together

Micrograph 5: Rubber particles of 10 % RSE with 2 parts soap. They were ruptured and attached to each other forming lumps.

Micrograph 6: Rubber particles of 15 % RSE with 2 parts soap. Note the broken rubber particles and its debris, present on the latex films

Micrograph 7: Immunogold–labelled rubber particles of the osmium tetroxide-fixed fresh field latex sample. The tiny black dots represent the 10 nm and 30 nm gold particles (some examples are pointed by the arrows)

Micrograph 8: Immunogold–labelled rubber particles of the osmium tetroxide-fixed HA latex concentrate sample

Micrograph 9: Immunogold–labelled rubber particles of the osmium tetroxide-fixed distilled latex sample. No gold particles observed

Micrograph 10: Immunogold–labelled rubber particles of the osmium tetroxide-fixed creamed latex sample. No gold particles observed