Anal. Chem. 2002, 74, 2541-2546
Elemental Mapping in Natural Rubber Latex Films by Electron Energy Loss Spectroscopy Associated with Transmission Electron Microscopy Ma´rcia Maria Rippel, Carlos Alberto Paula Leite, and Fernando Galembeck*
Instituto de Quı´mica, Universidade Estadual de Campinas, P.O. Box 6154, 13083-970, Campinas, SP, Brazil
Element distribution maps from Hevea brasiliensis natural rubber latex thin films were obtained, by electron energy-loss spectroscopic imaging in a low-energy (80 kV) transmission electron microscope. C, N, O, P, Na, Ca, Mg, Al, Si, and S maps are presented for latex fractionated by centrifugation, either followed by dialysis or not. Most elements forming non-carbon compounds are concentrated in small, electron-dense spots surrounded by a carbon-rich matrix of polymer, thus showing that the rubber is filled with small particles compatible with the polyisoprene matrix. Ca distribution is unique, since it closely parallels the C distribution, evidencing an important role for -COO--Ca2+-COO- ionic bridges in the structure of natural rubber. Natural rubber latex is a renewable polymer material displaying excellent physical and chemical properties and used in large quantities, ∼6 610 000 ton in 1998, by commercial, medical, transportation, and defense industries. Its global demand has been increasing at ∼200 000 ton‚year since 1993.1-3 It is extracted from the Hevea brasiliensis tree as a colloidal polydispersion in which negatively charged particles are suspended in a serum. The major particle groups are the rubber particles, the lutoids, and the Frey-Wyssling complexes.4 The particle diameter of rubber particles is between 5 and 3.000 nm, and they are surrounded by a complex misture of proteins, lipids, and long-chain fatty acids, which impart a negative charge to the particles. The rubber is soluble in many solvents, but there is usually an insoluble gel fraction, and the gel contents increase with storage, analogous to the storage hardening observed in the dry rubber.5,6 The rubber component is cis-1,4-polyisoprene, but the presence of carboxylate groups in the natural rubber was described by * To whom correspondence should be adressed. Tel: +55-19-3788-3080. Fax: +55-19-3788-3023. E-mail:
[email protected]. (1) Cyr, D. R. Natural Rubber. In Encyclopedia of Chemical Technology; KirkOthmer: New York, 1984; Vol. 20, p 468. (2) International Rubber Study Group (IRSG). Rubber Stat. Bull. 1999, 53. (3) Cornish, K.; Siler, D. J. Chemtech 1996, 26, 38-44. (4) Sethuraj, M. R.; Mathew, N. M. Natural Rubber: Biology, Cultivation and Technology; Elsevier Science: Amsterdam, 1992. (5) Tanaka, Y.; Tangpakdee, J. Rubber Chem. Technol. 1997, 70, 707-713. (6) Gazeley, K. F.; Gorton, A. D. T.; Pendle, T. D. Latex concentrates: properties and composition. In Natural Rubber Science and Technology; Roberts, A. D., Ed.; Oxford University: New York, 1988. 10.1021/ac0111661 CCC: $22.00 Published on Web 04/30/2002
© 2002 American Chemical Society
Burfield and Gan.7,8 Kawahara et al. demonstrated the presence of ester groups linked to the polyisoprene chain ends from both saturated (e.g., stearic) and unsaturated acids (e.g., oleic, linoleic, and linolenic).9 The former authors proposed carboxylate reaction with added cations (Ca2+) to form ionic cross-links responsible for storage hardening and leading to increased plasticity number and viscosity. Following this mechanism, Gan and Ting10 investigated the influence of main group mono- and divalent cations as well as transition metal ions on storage hardening due to reactions of carboxylate groups from the rubber chains. Na+ and K+ do not affect storage hardening10, but Ca2+ and Mg2+ reduce it, which is assigned to these ions blocking the carboxylate groups and preventing other reactions eventually leading to chain crosslinking. Other transition metal ions, such as Mn2+, Cu2+, and Fe2+, are catalysts of the oxidative degradation of solid rubber, to such an extent that the polyisoprene chain scission may become the main chemical reaction during rubber storage in the presence of increased amounts of these elements. Cu2+ ions are specially important, since copper compounds are used as latex production stimulants and fungicides. The effect of cations on the mechanical and adhesion properties of synthetic latexes was examined,11 specially those containing carboxylate groups.12-14 Rubber cross-linking by metal oxides and carbonates was described, in the case on butadiene-acrylonitrile,15 styrene-butadiene,16 and other synthetic polymers.17-21 Many elements are naturally found in the natural rubber latex, such as Ca, K, Al, Na, Mg, Mn, Fe, Si, Rb, P, N, S, and O, beyond C and H from the polyisoprene. Some of these elements are present within the lutoids, while others are in the serum. Many (7) Burfield, D. R.; Gan, S. N. Polymer 1977, 18, 607-611. (8) Burfield, D. R.; Gan, S. N. J. Polym. Sci. Polym. Chem. 1977, 15, 27212730. (9) Kawahara, S.; Kakubo, T.; Sakdapipanich, J. T.; Isono, Y.; Tanaka, Y. Polymer 2000, 41, 7483-7488. (10) Gan, S. N.; Ting, K. F. Polymer 1993, 34, 2142-2147. (11) Cooper, W. J. Polym. Sci. 1958, 28, 195-206. (12) Xu, Z. S.; Lu, G. H.; Cheng, S. Y. J. Appl. Polym. Sci. 1995, 56, 575-580. (13) Kim, J. H.; Park, Y. J. Colloids Surf. A 1999, 153, 583-590. (14) Chen, G.-N.; Chen, K.-N. J. Appl. Polym. Sci. 1999, 71, 903-913. (15) Matsuda, H.; Minoura, Y. J. Appl. Polym. Sci. 1979, 24, 811-826. (16) Sato, K. Rubber Chem. Technol. 1983, 56, 942-958. (17) Wiese, H.; Rupaner, R. Colloid Polym. Sci. 1999, 217, 372-375. (18) Huang, R. Y. M.; Wei, Y. J. Appl. Polym. Sci. 1994, 53, 179-185. (19) Ben Jar, P.-Y.; Wu, Y. S. Polymer 1997, 38, 2557-2560. (20) De, S. K.; Antony, P.; Bandyopadhyay, S. Polymer 2000, 41, 787-793. (21) Je´roˆme, R.; Moussaf, N. Polymer 1999, 40, 6831-6839.
Analytical Chemistry, Vol. 74, No. 11, June 1, 2002 2541
have well-defined biosynthetic roles, but some may just be absorbed from the soil into the sap.22-25 Neither latex coagulation nor centrifugation are able to fully remove the ions present, and significant amounts may end up within the solid rubber. This may account for observed variations in the mechanical properties of the latex, as well as for the uniqueness of the natural rubber, as compared to any synthetic rubber including cis-1,4-polyisoprene. The improvement of natural rubber mechanical properties by many inorganic additives is widely used in industry.26-28 Previous work from this laboratory revealed microchemical information on the distribution of chemical constituents within synthetic latex particle populations and individual particles, by using a combination of microscopies: energy-loss spectroscopy imaging in the transmission electron microscope (ESI-TEM), backscattered electron imaging in the scanning electron microscope (BEI-SEM) and scanning electron potential microscopy (SEPM) in the scanning probe microscope.29-35 This information proved very useful for understanding latex film-forming properties; generally speaking and following Cooke,36 microanalysis of industrial, complex products requires great attention. We have now applied multielement detection and mapping techniques to the study of natural rubber latex. In this work, we present ESI-TEM elemental distribution maps for natural rubber latex thin films. EXPERIMENTAL SECTION Sample Preparation. Latex was collected from RRIM 600 clone trees at the Instituto Agronoˆmico de Campinas and stored at 5 °C for a maximum 5 h. The latex was centrifuged at 10 000 rpm for 2 h in a Sorvall RC 26 Plus (Du Pont) centrifuge, and the upper fraction containing the rubber particles (RP) was redispersed in deionized water at 2.6% solids content. To remove micromolecular solutes, part of the RP dispersion was dialyzed against deionized water with daily changes for one week, within a SpectraPor regenerated cellulose membrane with a 3500 Da cutoff. Prior to use, the membrane was rinsed in deionized water at room temperature for 30 min, to remove the plasticizer. The dialyzed latex is designated as DRP (dialyzed rubber particles). The films for transmission electron microscopy were prepared by drying a droplet of RP and DRP suspensions on carbon-coated (22) d′Auzac, J.; Jacob, J.-L.; Chrestin, H. Physiology of Rubber Tree Latex; CRC Press: Boca Raton, FL, 1989. (23) Southorn, S. A.; Yip, E. J. Rubber Res. Inst. Malaya 1968, 20, 201-215. (24) Webster, C. C.; Baulkwill, W. J. Rubber; Longman: New York, 1989. (25) Verhaar, G. Processing of Natural Rubber; Agricultural Services Bulletin: Amsterdam, 1973. (26) Tokai Rubber Ind. Ltd. Japan Patent 11,315,165-A, 1999. (27) Bridgestone Corp. Japan Patent11,349,731-A, 2000. (28) Bando Chem. Ind. Ltd. Japan Patent 2,000,001,570-A, 2000. (29) Cardoso, A. H.; Leite, C. A. P.; Galembeck, F. Langmuir 1998, 14, 31873194. (30) Cardoso, A. H.; Leite, C. A. P.; Galembeck, F. Langmuir 1999, 15, 44474453. (31) Teixeira-Neto, E.; Leite, C. A. P.; Cardoso, A. H.; Silva, M. C. V.; Braga, M.; Galembeck, F. J. Colloid Interface Sci. 2000, 231, 182-189. (32) Amalvy J. I.; Asua J. M.; Leite, C. A. P.; Galembeck, F. Polymer 2001, 42, 2479-2489. (33) Galembeck, A.; Costa, C. A. R.; da Silva, M. C. V. M.; Souza, E. F.; Galembeck F. Polymer 2001, 42, 4845-4851. (34) Cardoso, A. H.; Leite, C. A. P.; Galembeck, F. Colloids Surf. A 2001, 181, 49-55. (35) Braga, M.; Costa, C. A. R.; Leite, C. A. P.; Galembeck, F. J. Phys. Chem. B 2001, 105, 3005-3011. (36) Cooke, P. M. Anal. Chem. 2000, 72, 169R-188R.
2542 Analytical Chemistry, Vol. 74, No. 11, June 1, 2002
Figure 1. Bright-field and elemental images of natural rubber latex film. The average film thickness is 45 nm. Scale bar is 1000 nm.
parlodion films supported in 400-mesh copper grids (Ted Pella). Procedure. The morphology and elemental distribution in the films were determined in a Carl Zeiss CEM 902 transmission electron microscope, equipped with a Castaing-Henry energy filter spectrometer within the column. When the electron beam passes through the sample, interaction with electrons from the different elements results in characteristic energy losses. The spectrometer uses inelastic scattered electrons to form element-specific images. A detailed description of the phenomena involved is in ref 30. Elemental images were observed for many elements found in this sample, using the monochromatic electrons with an energyselecting slit of 10 eV for carbon and 15 eV for the other elements. The energy-selecting slit was set at 303 eV for C, 410 eV for N, 544 eV for O, 358 eV for Ca, 1587 eV Al, 1871 eV for Si, 180 eV for S, 145 eV for P, 945 eV for Cu, 720 eV for Fe, 1325 eV for Mg, 650 eV for Mn, and 1090 eV for Na. The images were recorded using a Proscan high-speed slow-scan CCD camera and digitized (1024 × 1024 pixels, 8 bits) using the AnalySis software. Further image processing was done using the softwares ESI 3.0 and ACDSee. To make sure that the images presented are typical of the samples examined, six samples were prepared and visually examined to identify the existing patterns. At least three fields were examined for each sample, to make sure that the fields chosen for mapping were representative of the overall sample. Elemental images were acquired at least twice, for a given element
Figure 2. Bright-field and elemental images of a particle limited by the small black square in the bright-field image in Figure 1. Scale bar is 75 nm.
in a given sample. Whenever possible, many elemental maps were acquired from the same field. However, it was never possible to map every interesting element in the same field, due to sample damage by the electron beam. Every elemental map acquisition was preceded by recording the electron energy-loss spectrum for the element under investigation. To make sure that sample thickness was adequate for elemental mapping, the image contrast inversion was monitored, as the energy loss of the electrons used for energy-filtered imaging was changed from 0 to 20 eV. RESULTS AND DISCUSSION Film Prepared Using Rubber Particles. A bright-field picture of a dry rubber particle suspension film and many elemental maps of this same field are in Figure 1. Magnified views of the areas marked with squares in Figure 1 (bright-field image) are in Figures 2 and 3. Some darker points in the bright-field image appear bright in the elemental maps, evidencing that these are thicker particles, containing many elements. Carbon is easily mapped, since it is the predominant element. It accumulates in some areas, in good correspondence with the darker spots in the bright-field picture, but it is excluded from other areas. Ca distribution is well correlated with C, showing that this element is dispersed throughout the rubber matrix. N and O are broadly distributed, without the sharper accumulation spots observed, for
Figure 3. Bright-field and elemental images of the particle within the large black square in the bright-field image in Figure 1. Scale bar is 115 nm.
example, in the Al and Si (not shown) maps, which closely resemble each other. Fe and Mn maps (not shown) are also similar and they resemble the O map, while Na distribution follows the same pattern as Ca, and Mg follows P, concentrating in the denser particles. S and Cu (not shown) distributions are also similar, bearing some analogy to N but concentrating around the denser particles. These observations are consistent with the following picture: the rubber latex submonolayer is formed by a percolating net of fine and partly coalesced rubber particles, enclosing larger inorganic particles. There are also detectable nonparticulate constituents from the serum solutes, which are hardly noticed from the bright-field picture but appear clearly in the N and O maps and also (but less pronounced) in the C and Ca maps. The similarity between C and Ca patterns shows that Ca2+ ions are dispersed throughout the rubber phase, probably associated with carboxylate groups. Si and Al concentrate in the dense particles, probably as some sort of aluminum silicate or aluminum adsorbed on silica, but Fe, Mn, Na, and Mg are excluded therefrom, showing some accumulation at the particle borders. P, N, and S are mostly excluded from the particles’ bulk but they concentrate at their borders, what is consistent with these elements being associated with proteins and phospholipids. These compounds have amphiphilic properties, and they are thus prone to accumulate at the rubber-inorganic particle interfaces, sequestering other elements, such as Cu, that are easily complexed by proteins. Analytical Chemistry, Vol. 74, No. 11, June 1, 2002
2543
Figure 4. Bright-field and elemental images of dialyzed rubber latex film. The average film thickness is 63 nm. Scale bar is 1000 nm.
The enlarged particles presented in Figures 2 and 3 have different morphologies, and the first is more uniformly dense than the other. Films Prepared Using Dialyzed Latex. In this case, the spectral intensities for most elements beyond carbon were significantly less than in the nondialyzed latex, evidencing the loss of ions and small molecules during dialysis. Even so, many elemental maps could still be obtained, as shown in Figure 4. In the bright-field image, we observe small dark spots dispersed in an uneven background, showing that the film is very thin, nonuniform, and filled with particles. Following the elemental maps, carbon is dispersed throughout the film as expected, considering the rubber content, but concentrating at the same areas appearing darker gray (or cloudy) in the bright-field micrograph. On the other hand, the darker spots in the brightfield appear black but surrounded by bright halos in the carbon map, showing the low concentration of organic matter within the dense particles and its accumulation around them. The Al, S, Si, and P elemental maps are very similar (for which reason the latter three are only presented in the Supporting Information to this paper), and they show that the dense, small particles accumulate these elements. Oxygen is found in the small particles and also throughout the film, as well as N. However, spots with a marked depletion of N coincident with the dark spots in the C map are not seen in the O map. Calcium distribution is unique: this element is broadly distributed together with C but with a few accumulation spots. 2544 Analytical Chemistry, Vol. 74, No. 11, June 1, 2002
Figure 5. Bright-field and elemental images of the particles within the large black square in the bright-field image in Figure 4. Scale bar is 200 nm.
However, these do not correspond closely to the Al-, S-, P-, and Si-rich spots. Figures 5-7 are enlarged views of some areas from the same field, marked by the square and rectangles in the bright-field image within Figure 4. They confirm the previous observations but also provide some new information. First, the match between P, Al, and S bright spots within the denser particles is far from perfect, showing that these particles have a complex and nonuniform chemical composition. For instance, we observe the voids within some particles in the P and Al maps, which do not appear in the S maps. Moreover, these dark voids contain bright inclusions in the C map, showing that some organic matter is occluded by the dense particles. We can also observe the dispersion of Al outside the dense particles and throughout the film, more pronounced than P. Putting together this information, we can arrive at a concise description for the natural rubber latex film: polyisoprene is intimately mixed with N, O compounds and Ca, and complex, nonuniform inorganic particles are dispersed throughout. Considering that the nitrogen compounds are likely proteins, and observing the C and N accumulation evidenced by the bright rings around the dense particles, we can also state that the particles strongly adsorb organic film constituents, which can make a contribution to the latex film’s mechanical properties. The compability between the C-rich film-forming material and the particles dispersed throughout is very high, as evidenced by accumulation
Figure 6. Bright-field and elemental images of the particles limited by the middle black rectangle in the bright-field image in Figure 4. Scale bar is 150 nm.
Figure 7. Bright-field and elemental images of the particles limited by the small black rectangle in the bright-field image in Figure 4. Scale bar is 150 nm.
of organic matter at the particle interfaces and the interpenetration of film and particle constituents. This compatibility also explains why the inorganic particles were not separated from the rubber during centrifugation. The pattern of elemental distribution of the raw latex is probably the most relevant for the understanding of natural rubber latex properties, because the mapped elements are retained together with the rubber in most industrial processing procedures. Comparison of Ca and Al elemental maps shows a large difference in the abilities of these two elements to bind to the rubber matrix. This binding is likely made through COO--Ca2+COO- bridges, which are observed in many other ionomeric and polyelectrolyte polymer systems. Ionomeric structures can have an important role in determining rubber stability and mechanical properties, and they can obviously be modified, by adding other cations able to compete with calcium. Many rubber-processing procedures use zinc compounds.26,37 Tin,38 cobalt,28,39 lead,28 nickel,39 and iron39 compounds are also used but in special cases. Tanaka and Tangpakdee5 assigned the formation of a gel fraction in the natural rubber latex to intermolecular bridging by H-bonded proteins, as well as to cross-linking by phosphate esters and clustered long-chain fatty esters. Gel content may reach 50% after three-months storage, and different solubilizing amounts are
required for different solvents, suggesting that the gel cross-links are not all formed by covalent bonds.6 Calcium ion retention under dialysis (when most other cationic elements are lost) and its distribution throughout the rubber is consistent with its participation from the rubber structure, probably in ionic bridges with carboxylate ions. Since the carboxylatecalcium ion bonds are labile, this explains gel formation even in the absence of covalent bond formation. Mg ions do not seem to play the same role, since this element is not distributed throughout the rubber. This observation is consistent with the usual practice of coagulating NRL with calcium salts rather than any other salts, in the making of rubber gloves, and it points toward a high specificity in the interactions of these ions with the natural rubber. The specificity of the ion-polymer interactions observed in this work can probably be extrapolated to other polymer systems, specially synthetic latexes and polymers carrying carboxylate groups or their precursors, such as polyacrylonitriles, polyurethanes, polyesters, and polyamides.
(37) Peng, W.; Zhou, Z. China Patent 1,230,565-A, 2000. (38) Bridgestone Corp. Japan Patent 1,1349,731-A, 2000. (39) Gu, W.; Guo, G. Taiwan Patent 354,797-A, 1999.
CONCLUSIONS It is possible to map element distribution in a complex natural material, using low-energy (80 kV) electrons and very thin samples. Calcium ion maps show that this element has a unique behavior in the natural rubber latex, as compared to many other naturally occurring cations. Multielement inorganic particles are dispersed in rubber and the rubber-particle interfaces accumulate Analytical Chemistry, Vol. 74, No. 11, June 1, 2002
2545
nonrubber organic compounds, especially proteins, that make the particles compatible with the rubber, which is thus mechanically reinforced.
ACKNOWLEDGMENT The authors acknowledge grants from FAPESP, CNPq, and Pronex/Finep/MCT. M.M.R. is a Fapesp predoctoral fellow.
2546
Analytical Chemistry, Vol. 74, No. 11, June 1, 2002
SUPPORTING INFORMATION AVAILABLE Elemental distribution maps of elements cited in this paper but not shown in the figures. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review November 9, 2001. Accepted February 5, 2002. AC0111661
