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INDUSTRIAL AND ENGINEEHING CHEMISTRY
Formulas B and E are used with a slight addition of active black, but not enough to make sections 30 to 50 microns realized with the microtome definitely opaque. By making a slab similar to that of Figure 1, A and B, with such a compound, and cutting a section across a line of overflow (Figure 7), a much more complicated structure results instead of the thin luminous brush seen in Figure 1, C; the structure now completely covers the entire area of the microtome section. Still more revealing is a section taken from the overflow beyond the opening (Figure 8 ) . It is not necessary to confirm that in normal light the sections are completely uniform, apart from some parallel marks of the microtome blade. The small injection-molded specimen which, when made with Compound C (Figure 5 ) , indicated the oriented particles of talc shows a finer and more detailed structure when made with Compound B (Figure 9), giving a better idea of the complex movements of the injected mass. When tension stress-strain tests are carried out on 44.5/52.5mm. Schopper rings, they are cut from disks 57 mm. in diameter. The overflow of such a vulcanized disk, made with Compound B or E, shows in section a strong double refraction, which in fact originated these examinations (Figure 10). The flow of rubber toward the overflow is, however, easily seen in the peripheral regions of the disk (Figure 11, A ) . It also spreads inside the ring, which is punched with double circular knives and has not a very square outline; a section is shown in Figure 11, B. The tension stress-strain test is therefore made on specimens which are clearly anisotropic, in the case of the compounds examined ; but the optical criterion is perhaps excessively sensitive. All blacks give rise to this phenomenon, although to a lesser degree when the diameter of the particles is greater-for example, lampblack is less suitable than active or semiactive blacks. Red iron oxide, however, also behaves in the same way, with some dichroitic effect. When dealing with other white ingredients the actual double refraction of t,hc part.icles, or the repeated refractions of light, disturb the phenomenon. The characteristic anisotropy visible in misings containing a slight percentage of black is not confined to natural rubber. mixes. The behavior of synthetic elastomers, such as chloroprene, styrene, nitrile, and polgisobut~ylenel,ubbers, is similar.
Vol. 43, No. 2
I t is difficult to explain why such a small percentage of a h e pigment reveals a double refraction even when there are very slight movements. It can be supposed that the relaxation of the deformed rubber molecules is slower when very h e particles are dispersed between them, or that there is an orientation of the chains on which the structure of a carbon black depends. Even. if the second hypothesis is true, the practical and theoretical interest of the examination is not lessened, and it is only to be regretted that an equally simple examination of compounds having a high percentage of carbon black, where the effect should be even greater, is not possible. The introduction of talc has made it possible to note a lack of uniformity in the orientation of the particles of calendered sheets, but if 1% of carbon black is added, a much fmer and more complex structure, predominantly stratified, as in Figure 12, can be observed in calendered vulcanized sheets. The flows of rubber along the surface of the two cylinders (perhaps a t different temperatures), with a partial return of material when they join together to form the sheet, cause a complex distribution of shear rates leaving clear traces when observed under polarized light, We do not yet knoF, then, even in t,he simple case of calendered sheets, when it will be possible to find, not an isotropic, but a uniformly anisotropic material.
A ckn~w~ledgment Grateful acknowledgment is made to Luigi Emanueli, managiiig director of the Pirelli Co., for permission to publish thie paper.
Literature Cited (1) Ames, I n d i a Rubber J . , 67, 344 (1924). ( 2 ) Barwell, F. T., I n d e x Aero, 6, No. 1. 89 (1950). ( 3 ) Dawson, T. R., and Porritt, B. D., “Rubber, Physical and Chemical Prooerties.” D. 328. Crovdon. Enelsnd. . Reaearch Assoo. I
”
Brit. Ribber Manufacturers, 1935. (4) Gurney and Gough, Trans. I n s t . Rubber Ind., 22, 132 (1946). (5) Hurry and Chalmers, I n d i a Rubber W o ? l d ,119, No. 6, 717 (1949). ( 6 ) Kruse, J., Kollotd Z.,11, 100 (November 1948). (7) Scott, A. H., J . Reseurch Nntl. B u r . Standurds, 43, 355 (1949). (8) Smith, E. F., and IYangsgard, A . E’., J . Polymer Sci., 5, 168 (1950). (9) Van Rossem, India Rubber J . , 63, 343 (1921). RECEI>.I:DOctober 3, 1950
ic AND ~ ~ ~ ~ s - P B L Y I S Q P R E N FRO ES PLANT SPECIES
A SINGLE
Walter Schlesingsr and H. M. Eeeper Wm. Wrigleg dr. Co., Chicago, Ill. T h i s work was begun as part of a long-range fundamental investigation of the chemical and physical structure of chicle and other plant materials useful in the manufacture of chewing gum. The authors succeeded in demonstrating the existence of two types of hydrocarbon polymers in chicle and in identifying these with gutta (trans-) and caoutchouc (cis-) polyisoprenes from gutta-percha and Hevea rubber, respectively. Identification was accomplished by comparison of x-ray diffraction patterns and infrared absorption spectra of commercial chicle, and of a specially prepared sample of chicle obtained by tapping a single tree, with polymers isolated from gutta-percha Pahang and Hevea pale crepe.
This is the first proved example of the production of both cis- and trans-polyisoprene by a single plant. I t ie hoped that further study of polyisoprene isomerism in chicle and other substances will shed light on the important problem of the biogenesis of rubber and guttapercha.
niolecular weight polyisoprenes, as found in nature,
HIoccur ”” in two distinct forms, which are believed to differ from each other in the spatial configurations of the carbon atoms around the double bonds (1, 4,6). These two polymeric unsaturated hydrocarbons are exemplified by caoutchouc (from natural rubber), considered to be the cis- form, and by gutta (from gutta-percha or balata), hfJlicved to be the trans- form
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INDUSTRIAL AND ENGINEERING CHEMISTRY
399
L
Figure 1. Cylindrical Film X-Ray Diagrams of Guttas from Gutta-percha and Chicle t
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a. 6.
Gutta from gutta-percha. Pattern b Armour Researob Foundation Gutta from ahicle. Pattern by U. 9. Lubber Co.
The two forms are very different in physical appearance a t room temperature, the caoutchouc being amorphous and elastic and the gutta, crystalline and horny. Recently, a publication reported an unsuccessful attempt to find both caoutchouc and gutta in material obtained from the eame plant source (9). Saunders and Smith (8),although not specifically attempting to find both types of polymer, reported that chicle contains only the isomer corresponding to gutta. Studies in this laboratory have indicated the existence of both types of the hydrocarbon in chicle. The commercial product known as chicle is the concentrated latex of the sapodilla tree, Sapota achras (synonyms, Sapota zapotilla and Achras sapota). True chicle is obtained only from the area of the Yucatan peninsula, comprising Guatemala, British Honduras, and the southeasternmost tip of Mexico, although botanically related trees give chiclelike gums, which are sold under various names-chiquibul, nispero, crown gum, guaite, chicle faisan, etc. As a consequence of fairly careful supervision and control of the gathering and concentrating operations, the term “chicle,” today, describes a product of fairly constant botanical purity and quality. The trees are tapped during the rainy season (October through February) by itinerant tappers, called chicleros, who locate chicle trees, make a zigzag series of cuts about 18inches apart in the bark, and collect the exuded latex. The latex is boiled in an open vessel with constant stirring to reduce the water content to below 40%. The concentrated latex is then poured into molds to form, on cooling, blocks of about 20 to 25 pounds each. It is in the form of these blocks that commercial chicle is received in this country. Most of the work reported in this paper was conducted on chicle selected from commercial shipments from area8 which were known to produce chicle of high quality, relatively free from contamination and adulteration. I n order to verify some of the somewhat surprising results obtained, one sample of chicle latex was taken from a single tree and boiled down especially for this investigation. In a preliminary purification procedure, blocks of commercial chicle were melted in a steam-jacketed kettle and strained through a plate with 1/16-inch holes to remove large particles of bark, stone, etc. The still molten, strained chicle was centrifuged to remove fine grit and poured into pans to solidify. Chicle treated in this manner required several days’ standing at 20” to 25’ C . to “set” or crystallize sufficiently so that it could be chopped up into small lumps with hammer and chisel. During the heating, straining, and centrifuging the chicle changed color from almost white to a chocolate brown; and the water content was simultaneously reduced t o about 3 to lo%, depending on the water content of the crude chicle. (The sample of single-
tree chicle seemed entirely free of foreign matter and ww analyzed without this preliminary purification.) Commercial chicle used was from three sources. Commercial chicle I was from the southern portion of the producing area (Guatemala). Commercial chicles I1 and I11 were from west and east coastal areas, respectively, of the Yucatan peninsula in Mexico. The small sample, designated ST,obtained by tapping a single tree, was from British Honduras. One procedure for isolating the polymers from chicle has already been reported (9). In this scheme, Procedure A, inorganic
Table I.
a
Polymer Present in Chicle from Various Sources
(Calculated to dry weight basis) Crystalline Polymer (Gutta), % 12.4 Commercial chicle I 11.3 Commercial chicle I1 10.4 Commercial chicle 111 12.5 ST chicle Before aoetone extraction.
Table 11.
Av. H, %
Av.
Table 111.
Elementary Analysis of Chicle Polymers Crystalline Polymer (Gutta) 87.99 88.07 87.75 88.06 87.97 12.12 11.80 11.91 12.35 12.04
c, %
Noncrystalline Pol mer (Caoutoxouc), % 4.8’ 4.0 5.0 3.5
Noncrystalline Polymer (Caoutchouc) 87.91 87.79 88.04
Calculated for (C6Hs)n
87.91 11.77 11.81 11.81
88.16
11.80
11.84
Chemical and Physical Properties of Polymers from Chicle
Appearance Molecular weight (osmometric) No. of carbon atoms per double bond
Bromine No.
Iodine No. Melting point (capillary) O C. Hi h mdlting modi#cation. Low melting modification
Crystalline Polymer (Gutta) White, free-flowing, crystalline powder 16,000 4.90 6.10 69-71 64-66
Noncrystalline Polymer (Caoutchouc) Viscous, resinous, pale yellow t o colorless 91,000
...
6.05
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INDUSTRIAL AND ENGINEERING CHEMISTRY
Figure 2.
Flat Film X-Ray Diagrams of Caou-
tchoucs from Hevea Rubber and Chicle
a.
b.
Stretched, frozen Hevea rubber. Pattern by Armour Research Foundation Unstretched, frozen caoutchouc from chicle. Pattern by U. S. Rubber Co.
constituents and carbohydrates are aeparatnd from the chicl[a hydrocarbons and resins by dissolving the chicle in benzene at 25" to 35" C. The clear benzene solution, separated by centrifuging, is treated with ethyl acetate to precipitate gutta. The mot1it:r liquor obtained after filtration of the gutta is then treated with acetone, which precipitates the caoutchouc. An alternative scheme, Procedure B, has been found useful in examining a number of small samples of natural gums arid resins of diverse origin. I n this, the chicle is first extracted for 24 hours in the Soxhlet apparatus with acetone to remove resins. The resin-free residue is treated with hexane or petroleum ether at about 10" C. to dissolve caoutchouc. The material insoluble in
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4.0
5.0
6.0
7.0
8.0
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cold hexane is treated with benzene to dissolve gutta. The two polymers may be precipitated from the hexane and benzene solutions, respectively, with acetone or methanol. Yields of the two tlpra of polymers from several difiererlt batches of chicle are indicated in Table I. Yields reported ai(. those obtained by means of Procedure A, and are yields of acc tone-extracted polymers, except as otherwise noted. Result 5 of carbon and hydrogen determinations on the two polymers may be found in Table 11. These results were obtained 011 polymers isolated from t'he commercial chicles. Corresponding polymers isolated from commercial chicles 1, 11, arid TIJ wcr(a combined and the mixtures were analyzed. Table I11 cont'ains a brief summary of other physical m(1 chemical data on these polymers. Melting point data. are prcasented for high and low melting modifications of chicle guttii. These are believed, but have not yet been proved, to correspond to the alpha- and beta- modifications, respectively, of y u t h from gutta-percha. The free flowing crystalline appearance of chicle gutta as contrasted with the horny character of guttiL from gutta-percha and balata is believed attributable to differences in molecular weight. Chicle caoutchouc has the appearance of tt well masticated pale crepe. The polymers isolated from chicle were identified by cornparison of t'he x-ray diffraction patterns and infrared absorptioli spectra of these polymers with those of corresponding polymers isolated from Hevea pale crepe and gutta-percha (Pahang). In Figure 1 are shown the cylindrical film x-ray diagrams or alpha-gutta from gutta-percha and gutta from chicle. T h c chicle gutta pattern was obtained from the gutta isolated from the sample of chicle which was tapped from a single tree. The flat film x-ray diffraction patterns shown in Figure 2 provide :L comparison between Hevea pale crepe and the caoutchouc ohtained from single-tree chicle. The Hevea pat>ternshows pi'eferred orientation of crystals, inasmuch as the sample was both frozen and stretched. The chicle caoutchouc sample could not be stretched appreciably because of its relatively low rnolecular weight, and the pattern show11 is typical of an unstret,ched, frozwi cis-polyisoprene.
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10.0
11.0
12.0
13.0
14.0
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WAVE LENGTH, U , Figure 3.
Tnfrared Absorption Spectra of Chicle Gutta Compared with Those o f Hydrocarbons from Ilevea and Gutta-percha
\
I
D
Figure 4.
e
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INDUSTRIAL AND ENGINEERING CHEMISTRY
February 1951
GUTTA PERCHA
(p)( 11 1
U J i l l l 1 1 1 1 1 1 1 I I I I 1 1 1 1 1 1 1 1 1111 1 1 1 1 I l l I I I I I I I O , 4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
3.0
14.0
I
WAVE LENGTH, U , Infrared Absorption Spectra of Chicle Caoutchouc Compared with Those of Hydrocarbonn from Hevea and Gutta-percha
In Figure 3 are plotted the infrared absorption spectrum of the crystalline, gutta-type polymer from commercial chicle and the spectra of polymers obtained from Hevea rubber (pale crepe) and gutta-percha Pahang, respectively. In Figure 4 are shown the infrared absorption spectra of these same polymers from gutta-percha and pale crepe, together with the spectrum of the noncrystalline caoutchouc-type polymer from commercial chicle. These curves were made by tracing, without distortion, the original per cent transmission os. wave-length curves onto a single plot, arbitrarily separating the curves from each other in the vertiral direction to minimize confusing interlacing and to permit comparison of absorption maxima. The curves were traced in such a way that the same wave-length scale served for all the curves on each figure. General similarity between the absorption spectrum curves for chicle caoutchouc and the corresponding material from Hevea is seen. The close conformity of the chicle gutta curve with that of the polymer from gutta-percha Pahang may be noted. It is interesting to examine, in somewhat more detail, points of similarity and difference between gutta and caoutchouc in the light of previously published information on the same subject. Saunders and Smith (8) and Hendricks, Wildman, and Jones (8) have examined the infrared absorption spectra of these and similar polymers. I n both of the publications cited, the spectra in the region of 12 microns were considered to be characteristirally different for the two types of polymer. Saunders and Smith pointed out the feasibility of differentiating between alphagutta and beta-gutta by examining absorption spectra in the 12-micron region. They also indicate, by comparison of crystalline and amorphous caoutchouc with crystalline and amorphous gutta, spectral differences between these materials which are due to crystallinity alone and not cis-, trans- isomerism. The chicle gutta shown in Figure 3 corresponds closely to the betagutta-percha of Saunders and Smith with respect to the shape and intensity of the absorption bands a t 11.4, 12.5, and 13.3 microns. The strong absorption maximum a t 11.95 microns of the caoutchouc samples, and the slight shift and less intense
absorption for the gutta samples are in accordance with published data both of Saunders and Smith and of Hendricks and eo-workers. Preliminary studies of alpha-, beta- isomerism of gutta from chicle show that the low melting form is the one recovered when either form is dissolved in carbon tetrachloride or carbon disulfide and the solvent allowed to evaporate rapidly, as was done in casting films for these spectra. In addition to the differences in spectra in the 12-micron region, several other small but nevertheless significant differences between spectra for caoutchouc and gutta have been noted. Examination of the curves of Saunders and Smith and those of Hendricks and co-workers shows agreement with the authors’ curves a t several points. At 8.02 microns amorphous caoutchouc shows a broad absorption band which does not appear in the gutta curves. The data of Saunders and Smith show the spectrum of crystalline caoutchouc to differ from amorphous a t this point, but the spectra of both forms differ from gutta in this region. A strong absorption a t 8.27 microns for gutta is absent in amorphous caoutchouc, However, the curves of Saunders and Smith for crystalline caoutchouc reveal an absorption band at this wave length. At 8.72 microns gutta shows a strong absorption band which seems to be shifted to about 8.84 microns for caoutchouc, A sharp absorption maximum a t 9.07 microns for gutta has apparently been shifted to about 9.21 microns for caoutchouc and the maximum broadened somewhat. A small shift of a beta-gutta maximum at 9.69 to 9.64 microns for caoutchouc is apparently connected with differences in crystallinity, inasmuch as the data of Saunders and Smith show alpha-, beta-, and amorphous gutta to have different absorption spectra in this region. These points of difference appearing in three independently obtained infrared absorption spectra for gutta and caoutchouc do not seem to be the result of mere coincidence, but are believed to represent additional check points for differentiating these polymers. I n addition to these points of agreement between the authors and other workers, the authors’ curves show a few other points
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INDUSTRIAL AND ENGINEERING CHEMISTRY
which might be of use in the differentiation of gutta from caoutchouc by infrared spectra. This second group of points is considered separately because in some cases the absorption maxima are not too clearly defined and because they agree with some but not all of the previously published data. The authors' curves show a shift in an absorption maximum from 7.54 microns for gutta to 7.62 microns for caoutchouc. Two gutta absorption maxima a t 7.82 and 7.95 microns do not appear in the caoutchouc curves. However, beta-gutta only, and not alpha-, possesses these absorption maxima. An absorption peak at 10.04 microns for beta-gutta (or 10.11 for alpha-gutta) seems shifted to 9.90 microns for caoutchouc. A gutta absorption peak a t 10.40 microns does not appear in the amorphous caoutchouc curves, although the data of Saunders and Smith show crystalline caoutchouc to be like beta-gutta a t this point. Finally, a shift in an absorption maximum at 10.88 microns for gutta to 10.78 microns for caoutchouc appears in the authors' curves. The absorption data presented for gutta are for the beta modification, except as otherwise noted. Absorption spectra of the alpha modification show some differences between it and betagutta, particularly in the region 11.3 to 13.3 microns. On the basis of the authors' results there seems little doubt that rhicle contains a gutta-type hydrocarbon of low molecular weight as well as a caoutchouc hydrocarbon. This somewhat surprising result is confirmed by the separation of the two types of hydrocarbon from the sample of singletree chicle. Prochazka and Endemann ( 6 ) in their study of chicle found two substances, which they designated as cold ether-soluble and cold ether-insoluble hydrocarbons, respectively. Their elementary analyses, however, did not conclusively show their products to be hydrocarbons, and their work seems to have been ignored by later workers on chicle.
Experimental Separation of Polymers. PROCEDURE A. This method is best applicable when the quantity of starting material exceeds 50 grams. One hundred grams of chicle were treated with 150 ml. of thiophene-free benzene and allowed to stand for 24 hours a t 30" to 35" C. with occasional shaking to ensure complete dispersion. This mixture was then centrifuged to separate the transparent, yellow-brown benzene solution from the insolubles. To the benzene solution were then added, with stirring, 300 ml. of absolute ethyl acetate, and the mixture was refrigerated a t 5" to 10" C. oveiaight. The crude gutta which separated was filtered with suction on a Buchner funnel and rinsed twice with 10-ml. portions of ethyl acetate. This gutta was extracted in a Soxhlet apparatus with acetone for 24 hours to remove waxy and resinous impurities. The acetone-extracted material was then extracted for 24 hours in a Soxhlet apparatus with peroxide-free ethyl ether, The insoluble extracted gutta was air-dried to constant weight. Gutta obtained by this proced.ure was.found to melt a t about 68" to 71" C. (the melting point varylng slightly from sample to sample) and is believed to correspond to the alpha form of gutta from gutta-percha or balata. A lower melting form (melting point 64' to 66' C.) may be obtained by melting this gutta and cooling rapidly or by recrystallizing from petroleum ether or other solvents. The caoutchouc-type polymer was precipitated from the filtrate obtained hy filtering off the crude gutta, by addition of 450 ml. of acetone (containing about 5 m of sodium iodide to acetate solution. aid flocculation) to the clear benwne-dyl The precipitated polymer was recovered by decanting off the supernatant liquid, and, was purified by a 2Phour extraction in 8 Soxhlet extractor with acetone. It was then dissolved in benzene, filtered through paper, and reprecipitated with acetone. The precipitate was rinsed mth acetone and dried under vacuo a t room temperature.
PROCEDURE B. This separation scheme is best applied to Bamples of less than 60 grams. Ten grams of chicle were extracted with acetone for 24 hours in a. Soxhlet extraction apparatus. The insoluble material was
Vol. 43, No. 2
removed, allowed to air-dry, and immersed, while still in the extraction thimble, in I50 ml. of cold Skellysolve B in a refrigerator a t about 10' C. The mixture was allowed to stand for 48 hours, with occasional agitation, a t the end of which time the thimble was removed from the solvent and the contents were washed several times with fresh cold solvent. The combined Skellysolve B solutions were then treated with excess acetone and a few drops of concentrated aqueous sodium iodide solution were added to assist in the coagulation. After standing overnight in the refrigerator, the supernatant liquid was decanted and the preci itated caoutchouc was washed several times with acetone. TRe caoutchouc was dried in a vacuum desiccator overnight The Soxhlet thimble containing the material insoluble in dold Skellysolve B was placed in a wide-mouthed Erlenme er flask containing 150 ml. of benzene a t about 25" to 35" C. anJallowed to stand for 48 hours with occasional agitation. The gutta solution so obtained was clarified, if necessary, by centrifuging or filtration, after which operation the gutta was precipitated by addition of excess acetone or methanol. After standing overni ht in the refrigerator, the gutta was filtered and allowed to air-& a t room temperature Molecular Weight. Molecuiar weight was determined by the method of Sands and Johnson (7). As osmosis membranes, undried regenerated cellulose film, treated in accordance with directions given by Wagner (IO), was found to be satisfactory for both the caoutchouc and gutta polyisoprenes. Bromine Number. Bromine number was determined by reaction of a solution of polymer in carbon tetrachloride with acidified 0.1 N bromide-bromate solution for 4 minutes. Excess aqueous potassium iodide solution was added to the mixture, which was then back-titrated with sodium thiosulfate to the disapprarance of iodine color. Iodine Number. Iodine number as determined by the method of Lee, Kolthoff, and Mairs (3). X-Ray DifIraction Diagrams. The x-ray patterns (Figure 1) of the gutta samples were obtained a t room temperature by the conventional Debye-Scherrer method. The fiber diagram of the Hevea sample (Figure 2, a ) was obtained by stretching a strand of the material a t 0" C. The temperature was then reduced to -30" C. and the exposure was made. The diffraction pattern of the chicle caoutchouc sample (Figure 2, b ) was obtained by freezing the sample a t -27' C. for 48 hours and exposing it in the x-ray camera in the unstretched condition. Infrared Absorption Spectra. Infrared absorption spectra were determined with the Baird Associates, Inc., double-beam infrared spectrophotometer. Spectra were obtained on dry films prepared by dissolving the polymers in carbon tetrachloride or carbon disulfide, depositing the solutions on rock salt plates of 0.25-inch thickness, and evaporating the solvent. Film thickness varied slightly from sample to sample. This variation accounts for some of the quantitative differences noted in percentages of transmission.
Siinriiiary 1. Chicle has been shown to contain two polyisoprenes, widely different in physical appearance, but identical in chemical composition and degree of unsaturation. 2. By means of infrared absorption sprctra, one of these polyisoprenes, a white, crystalline powder, has been shown to correspond to the polymer of gutta-percha, and is presumed to be the trans- modification. The other polyisoprene, which has the appearance of well broken-down rubber, corresponds to Hevea rubber and is presumed to be the cis- modification of polyisoprene. 3. The two forms of polyisoprerie have been demonstrated to occur in approximately the same percentages in chicle from four different geographical sources, one of these chicle samples having been extracted from a single tree. 4. Two methods for isolating these polymers from commercial chicle have been presented and may be extended for use in the qualitative and quantitative determination of polyisoprene in any dried natural latex.
Acknowledgment The authors wish to acknowledge the advice and helpful criticisms given by H. W. Conner of the Wm. Wrigley Jr. Co. They are indebted also to P. C. Hutchinson and E. L. Flaherty of Baird Associates, Inc., for infrared absorption spectra, and to H. N. Campbell of U. S. Rubber Co. for x-ray diffraction data,
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INDUSTRIAL AND ENGINEERING CHEMISTRY
The kind cooperation of Stanley Siege1 and Irene Corvin of the Armour Research Foundation in obtaining a portion of the x-ray data is acknowledged.
Literature Cited (1) Bum, C. W., Rubber Chem. and Technol., 15,704 (1942). (2) Hendricks, 8. B., Wildman, S. G., and Jone8, E. J., Arch. Biochum., 7 , 427 (1945); Rubber Chem. 4 Technol., 19, 501 (1946). (3) Lee, T. S., Kolthoff, I. M., and Mairs, M. A., Zbid., 21, 835 (1948).
403
(4) Mark, H., “Physical Chemistry of Hlgh Polymeria Substanoes,” p. 175, New York, Interscience Publishers, 1940. ( 5 ) Meyer, K. and High Polymers,,, p. 119,New York, Intersoienoe Publishers, 1942. (6) Prochazka, G.A., and Endemann, H., Pharm. J . (3rd series), 9, 1045, 1067 (1879). (7) Sands, G . D.,and Johnson, B. L.,Anal. Chern., 19,261 (1947). ( 8 ) Saunders, R. A., and Smith, D. C., J . Applied Phys., 20, 953 (1949). (9) Schlesinger, W., and Leeper, H.M., Science, 112,51 (1960). (10) Wagner, R. H., IND.ENQ.CHEM.,ANAL.ED., 16,520 (1944). RECEIVED October 16, 1950.
Esterase in Latex of Hevea bradliQns8s I . N. S. De Haan-Homans IndonesZech InetZtuut Voor Rubberonderwek, Bogor, Indoneeia
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b
*
which naturally contain good T h e investigations described here were for the greater ITERATURE does not emulsifiers, by vigorously part carried out during the Japanese occupation of Java. mention the presence shaking i t by hand for 3 Because of shortages of materials at that time, it was imof an esterase in the latex of minutes in a small glasspossible to perform the experiments as elaborately as was Hevea braeilim‘s, but the stoppered Erlenmeyer flask. desirablefor example, ethyl and methyl butyrate were presence of a lipase in the At first, 2 to 5 ml. of a buffer unobtainable. seeds of Hevea brasiliensis has solution were added (acetate No fat-splitting enzyme-that is, no l i p a s e w a s found been proved (1,8). Furtherbuffer, but generally phosin the latex of Hevea brasiliensis. I t was proved, howmore lipases have been found phate buffer), but later on ever, that this latex contains an esterase which decomposes in the latices of other plants this was discontinued as latex, low esters (acetates above all and ethyl acetate in particu-for e x a m p l e , E u p h o r yellow fraction, or serum lar) rapidly and to a high degree. biaceae, Ficus carica, Maclura buffer is satisfactory. One Experiments carried out at the Indonesian Rubber ReMoms, Carica papaya (7, 8). drop of toluene was added to search Institute also demonstrated that the pH of the Gerber (2) studied the acthe emulsion to prevent baclatex, about 7 on exudation from the tree, decreases raptivity of enzymes, including terial activity. The contents idly immediately after exudation, and within a short time a lipase, in the latex of Browof one Erlenmeyer h s k of reaches 6.2 to 6.5. The esterase may be involved in this sonetia papyrifera. in the each series were examined process. Further work will be necessary to find an answer various seasons. immediately after emulsificato this question. When investigating the tion; the remaining flasks spontaneous coagulation of were incubated a t 37” C. for latex, v a n Gils ( 8 ) and periods of 0.5 to 48 hours and then examined. These were rinsed Altman arrived at the supposition that a fat-splitting enzyme with 25 or 50 ml. of alcohol, if necessary, and titrated with 0.1 N might be involved in the process. van Gils (4) succeeded in sodium hydroxide. I n control experiments, in which Ricinus oil accelerating the spontaneous coagulation by adding a prepwas investigated with pancreas lipase, the oil was prepared acaration of pancreas lipase to the latex. This lipase preparacording to Willstlltter (9) and emulsified in a buffer solution by tion had only been very roughly purified, however, and it shaking it with albumin. Other emulsifiers, such as Exanol, probably contained a large portion of the other pancreas enzymes, lissolamine and triethanolamine oleate were found to inhibit the such as maltase, amylase, proteases (trypsin), and possibly leciactivity of the enzymes too much. thinase. The two last-mentioned enzymes in particular may also There are difficulties attached to the use of latex and yellow have influenced the stability of the latex. The lipase promoting fraction as the rubber coagulates during shaking or when alcohol the spontaneow coagulation might be present naturally in the is added. Before titration the coagulum was squeezed out and latex, or it might be formed by growth of bacteria. It was consesubsequently removed. quently highly desirable to determine first of all whether the Inasmuch as the serum obtained by freezing gave the best latex of Hevea brasiliensis naturally contained a fat-splitting results, this serum or the yellow fraction was generally used. enzyme. By way of control, a few mixtures to which no substrate had been Methods added were made with each experiment (to determine the selfacidification of the latex or the serum) as well as emulsions withInvestigations concerning the presence of the enzyme were out enzyme solution or with boiled serum (to trace the autolysis carried out with fresh latex and with the components obtained of the substrate). from it, such as serum obtained by coagulating latex with acid (coagulation serum) or by freezing the latex and thawing i t after Experiments with 6 c L ~ tFat,” e ~ Ricin- Oil, 5 to 7 days (serum o.b.f.), the yellow fraction, and coagulation Arachis Oil, and Monobntyrin serum and serum by freezing from the yellow fraction. The If the assumption of van Gils and Altman that a lipase is inp H was increased by the addition of a small quantity of sodium volved in the spontaneous coagulation of latex is correct, the subhydroxide solution or decreased by a little sulfuric mid. strate for the lipase must also be present in the latex. The accurately weighed quantity of substrate (0.26 to 1 gram) Owing to the lack of good agents for the extraction of fats dur-. was emulsihd in 2 to 5 ml. of the components mentioned above,
