Microscopic tudies of Lyogels PREPARATION OF SAMPLES FOR ULTRA-ILLUMINATION BY INCIDENT LIGHT ERNST A. HAUSER Massuchusetts Institute of Technology, Cambridge, Muss.
D. S . LE BEAU Midwest Rubber Reclaiming Company, East S t . Louis, I l l .
A detailed description of the technique used in preparing samples for microscopic studies with ultra-illumination by incident light is given. The versatility of the method is discussed, and special attention is draw-n to the possibility and advantage of color microphotography and fluorescence microscopy. Differencesin the properties and in the molecular configuration of several high-molecular compounds are discussed on the basis of their microscopic morphology.
. T
HE use of ultra-illumination by incident light for microscopic studies of lyogels (3) offers such a wide applicability that a more detailed description of the technique uscd in preparing the specimens and a discussion of some new results and their significance seemed appropriate a t this time. Of all the ultramicroscopes, only the Ultropak fulfills the requirements, because it is the only instrument which permits circular illumination of the preparation by incident light without passing through the lenses of the objective. The light can also be focused independently of the focal distance of the optical system ( 3 ) . This permits
illumination of the preparation in a way best suited for its surface configuration and prevents blurring by light scattering. I n preparing sxnples of rubber for electron microscopy, the solution was deposited on a perforated nickel or platinum plate (1, 2, 6). When an attempt was made to use the same depositor for the microscopic studies with ultra-illumination by incident light, i t became evident that the light reflected from the surface of the depositor (Figure 1) made it difficult t o obtain clear pictures. I n addition, the contours of the open spaces were rough and did not permit good film formation. Therefore, a copper wire gauze with very small meshes (3)was selected (Figure 2). However, by calendering the gauze between two steel rolls until its thickness had been reduced to about one third of the original, a smooth sharp-edged surface was obtained (Figure 3). PREPARATION OF SAMPLE
Natural and synthetic rubber samples are first dissolved in small concentrations in an appropriate solvent, and this solution is spread on a nonmiscible liquid. Then the depositor is inserted below the solution a t a spot where it had not spread, and the de-
Figure l (Left). Perforated Nickel Plate ( X 1500)
Figure 2 ( R i g h t ) . Copper Wire Gauze ( X 2OOo)
Figure3 (Left). Calendered Copper Wire Gauze ( X 25UO)
Figure4 (Right).Pale Crepe Milled for 20 Minutes, Deposited from Benzene ( X 2500)
335
336
INDUSTRIAL AND ENGINEERING CHEMISTRY
Vol. 38, No. 3
example, polystyrene in styrene-it is advisable to lift the gauze through the solution immediately after it has been spread on the nonmiscible me@um t o avoid difficulties caused by evaporation of the solvent. From an optical point of view, best results are obtained if the film is deposited so that it forms on the top surface of the gauze (Figure 4). Then the preparation can be illuminated by light leaving the circular lens system of the objective a t a very flat angle which prevents the light from being reflected from the gauze before it reaches the specimen.
positor is lifted upward through it (3). Since the state of aggregation of natural and synthetic rubber in solution changes with time and temperature, it is advisable to make the specimen from the solutions a t a fixed time interval after the latter has been prepared whenever compargble studies are contemplated. However, the concentration and the viscosity of the solution, as well as its interfacial tension with the medium on which it is spread, must be taken into consideration. Therefore, it is impossible to lay down a strict rule as to how best results can be obtained, and the optimum conditions must be determined for every specimen. A few general suggestions may be of value. Solutions of capillary active substances like soaps preferably should not be deposited in the manner described (3)because this might produce an orientation of the molecules in the deposited film which would not be representative for the bulk of the system. Therefore, when working with such substances the gauze is dipped vertically into the solution, withdrawn very slowly, and allowed to dry before being placed on a microscope slide used as support. When working with synthetic resins dissolved in their monomer-for
COLOR MICROPHOTOGRAPHY AND FLUORESCENCE MICROSCOPY
Even more striking photomicrographs can be obtained by using color films or plates such as Kodachrome A. If the color slides are projected onto a screen or examined through a film viewer, the preparation can be studied a t very great magnification and a tridimensional effect is also apparent. Cylindrical or spherical structures and flat bands can be differentiated by this means.
5
8
(x 2500)
Figure 5.
GR-S Recovered from Benzene
Figure 6.
Methyl Methacrylate Recoverea nitrile (X 2 W )
Figure 7.
Electron Photomicrograph of Sodium Oleate (5)
from Acrylo-
Figure 8: Palmolive Soap (X MOO) Figure 9.
,
,L‘
i
Balata Recovered from Xylene ( X 1500)
March, 1946
INDUSTRIAL AND ENGINEERING CHEMISTRY
331
Figure 10. GR-S Recovered from Benzene (X 3000) .Figure 11. Hycar OS-10 Recovered from Benzene (X 4000) Figure 12. Hycar OR-25 Recovered from Benzene ( X 4000) Figure 13. Neoprene GN Recovered from Benzene (X 4000) Figure 14.
Styrene ( X 4000)
10 11
13
Figure 15. Withdrawal of Micromanipulator Needles ( X 1000) Figure 16. Crepe Sol Recovered from Benzene ( X 3000)
12
14
INDUSTRIAL AND ENGINEERING CHEMISTRY
338
Another interesting application is the use of ultraviolet radiation as a source of illumination; the lens system through which the light passes before impinging on the preparation must be made of quartz or glass which does not absorb ultraviolet rays. This technique will prove valuable in studying the distribution of compounding ingredients. Preliminary experiments have shown that it will be possible t o study the progress of vulcanization, because most of the commonly used accelerators exhibit pronounced fluorescence. It will also be possible to ascertain whether the entire high molecular substance fluoresces, whether this phenomenon is characteristic only of the low-molecular-weight fractions, or whether it results only from the fluorescence of retained solvent, It is known that a solvent can incite fluorescence by increasing the distances between the moleculcs of the solute so that they no longer interfere with their own oscillations ( 7 ) . This is proved by the fact that the intensity of fluorescence can be drcrcascd by increasing the concentration of the solute. HIGH POLYMERS AND FILM FORMATION
When certain synthetic poll mers are deposited-for example, GR-S or methyl methacrylate-the breaking of the original film (Figures 5 and 6) differs radically from that obtained from natural rubber. It has also been observed that aging of the preparation has a marked influence on the distribution and size of the globules in the case of natural rubber. The number of blobs definitely increases with time. The presence of globules in soap was shown in a previous publication ( 3 ) . However, if such a preparation is aged, the picture changes, the globules disappear, and structures identical with those reported by electron microscopy (1, 6) result (Figures 7 and 8). Great difficulties have been encountered in making preparations of certain GR-S samples, particularly those characterized by high gel content, and also of balata; both of these materials show no globules and very ragged film contours (Figure 9). These experiments, although not conclusive, may be considered the first visual proof of the difference in molecular structure between natural rubber and balata, as well as that of high-gel-content GR-S. Balata represents the trans configuration of polyisoprene, whereas rubber must be considered its cis configuration. The average chain length of balata does riot differ appreciably therefore, the difference in from that of guayule rubber (3,4, configuration of the molecules must be considered responsible for their different properties and also for the difficulty with
Vol. 38, No. 3
which threads are formed by balata. Present knowledge of the molecular coilfiguration of GR-S is not yet advanced enough to offer a similar explanation. However, a decided difference can be noticed in the morphology of low- and high-gel-content GR-S samples. The former show some very small globules but are mainly characterized by bandlike filaments (Figure IO). Hycar OS-10 (Figure 11) shows bands, filaments, and globules, although the latter are rather rare; Hycar OR-25 (Figure 12) shows only strong and netted bands. Neoprene GN (about 2 years old) reveals only a network of bands (Figure 13). Styrene (polymerized a t 90” C. for 48 hours without catalyst) shows extraordinarily stiff and breakable threads without actual netvork formation (Figure 14). Two observations should be recorded. If a string of droplets is obtained by depositing a solution of crepe rubber between two or three micronianipulator needles and then nioving them apart (Figure l5), the droplets elongate but rcappear upon release of the tension. This might indicate that elongation has caused the short molecules to align. That the droplets are composed of matter in a liquid state but of fairly high surface tension, or that the surface layer of the drops is composed of matter in an oriented coherent condition, is shown by some drops which have been pulled out of their alignment in the thread by being attached to another thread under tension (Figure 16). If that thread is destroyed, the drop immediately aligns itself in the remaining thread. The foregoing results indicate that a more detailed study of other plastics by this new technique will reveal some valuable facts pertaining to their structure; it is hoped that the detailed discussion of the preparation of samples will aid others in using this technique for their special problems. LITERATURE CITED
(1) .4nderson, T. F., in “Advances in Colloid Science”, 1’01. I, p. 353, New York, Interscience Publishers, 1942. (2) Hall, C. E., Hauser, E. A., le Beau, D. S., Schmitt, F. O., and Talalay, P., IND.ENQ.CHmr., 36, 634 (1944). (3) Hauser, E. A,, and le Beau, D. S., Ibid., 37, 786 (1945). (4) Hauser, E. A., and le Beau, D. S., India Rubber W o r l d , 107, 68 (1943); 108, 37 (1943). ( 5 ) Marton, L., MMcBain, J. W., and Vold, R. D., J. A m . Chem. Soc., 63, 1990 (1941). (6) Prebus, A. F., in Alexander’s “Colloid Chemistry”, Vol. V. p. 187, New York, Reinhold Pub. Carp., 1944. (7) Pringsheim, P., “Fluoreszenz und Phosphoreszenz im Lichte der
neueren Atomtheorie”, Berlin, Julius Springer, 1921.
Boiling Points of Three Isomeric Heptanes FRANK S . FAWCETT Socony-Vacuum Oil Company, Znc., Paulsboro, N. J . Boiling points of 2,2-dimethylpentaneY 2,e-dimethylpentane, and 2,2,3-trimethylbutane have been determined by the comparative dynamic method over the range 1-15 atmospheres, using n-heptane as the reference liquid.
I
N A CONSIDERATION of the separation by distillation a t
pressures above atmospheric of three paraffin hydrocarbons boiling within a narrow temperature range, vapor pressure data were obtained by the comparative dynamic method (5, 6), using metal Cottrell ebulliometers with glass pumps. Successive measurements were made of the boiling point of the substance
under investigation and of the boiling point of n-heptane in ebulliometers connected to the same reservoir in which the pressure could be varied. The data were observed as a series of corresponding boiling points of the substance and of n-heptane at the same pressure. The Washburn-Read modification ( 7 ) of the Cottrell ebylliometer (1) was used (Figure 1). The body of the ebulliometer consisted of a section of ll/c-inch standard seamless steel pipe; the diameter was reduced at the ends, and one end was sealed with a disk welded in place. The skirt surrounding glass pump D was of 1-inch seamless steel pipe turned down to l/,rinch wall thick-
