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V O L U M E 2 7 , N O . 11, N O V E M B E R 1 9 5 5 in aniline has been estimated from a cloud point with rapeseed oil or a cottonseed oil-heavy mineral oil composition and in furfural with a hexanol-cottonseed oil reagent; in textiles and dehydrated foods by equilibrium humidity; in ammonium nitrate by the boiling of the melt a t reduced pressure; in acetic acid by a polarimetric method; in gases by the absorption of the water in an organic solvent on which the conductance was determined; and in textiles, ceramics, paper, tobacco, and petroleum oils by electrical resistance. Chemical methods used include manometric ones, such as the measurement of hydrogen evolved after treatment with sodium; titrimetric methods, such as the reaction between water and magnesium nitride to give ammonia, which is absorbed and titrated with sulfuric acid; the sodium ester method for small quantities of water in alcohol; hydrolysis of acetyl chloride in the presence of pyridine, which detects 0.02y0 water but is subject to much interference: and anhydrous cupric sulfate and potassium permanganate. The most widely used method for the determination of small quantities of water is the Karl Fischer, which uses a solution of iodine, sulfur dioxide, and pyridine in methanol. This method has a sensitivity as low as 0.005% with an accuracy of within i O . O O l % . Here a simple and very sensitive method, which makes use of the extreme solubility of methylene blue in water, has been developed in connection with the determination of moisture in hexachloroethane, which has a limit of 0.05% rater. One volume of the sample is dissolved in two volumes of carbon tetrachloride, technical grade, Octagon Process Inc., and a small quantity (approximately 10 mg.) of dry methylene blue, water-soluble, technical grade, Fisher Catalog KO. A-766 is added. Water present in the sample tends to be released owing to decreased solubility in the mixture of hexachlorethane and carbon tetrachloride, and coagulates in globules. In the globules, the methylene blue, which remains as a reddish brown powder on the surface of the carbon tetrachloride, turns a deep blue,
indicating the presence of the water. This indication appears with as little as 0.03 mg. of water, although when the water content goes below 0.1 mg. the globules may not appear on the surface, but may adhere to the sides of the beaker, in which case vigorous stirring and policing are required to disperse the methylene blue. The sensitivity was determined by using samples of a jet fuel, (Navy Grade JP-5, or heavy end aviation fuel, HEAF), saturated with water. The saturation point of this jet fuel had been carefully studied in this laboratory and determined to be at 0.011% water by means of the classical Karl Fischer method. Various volumes of this water-saturated jet fuel were dissolved in carbon tetrachloride and methylene blue was added. As little as 0.08 mg. of water, representing 0.000016% on the basis of a 500-gram sample, was indicated by the methylene blue. On a sample of turbine oil as little as 0.03 mg. of water was detected. The dry jet fuel when dissolved in the carbon tetrachloride gave no reaction with the methylene blue. Because this technical grade of carbon tetrachloride showed no reaction at all with the methylene blue, it was assumed to be anhydrous. It is believed that this method could be made applicable to other substances with only traces of water, so long as the substance itself gives no reaction with methylene blue. Both dry acetone and dry benzene give no reaction xith methylene blue, and thus may be used also as media. I t is possible to develop strips, made of materials inert to methylene blue and impregnated with dry methylene blue, which detect traces of water on dipping. This method, of which systematic investigation is under way a t this laboratory is applicable also to moisture detection in gases. 164 Hart St. Brooklyn 6, N. Y .
FLORENCE h-ESH
The opinions or assertions herein are those of the author, and are not construed as reflecting the views of the Navy Department or the Naval Service a t large.
MEETING REPORT
Symposium on J O H N W. SHELL The Upjohn Co., Kalamazoo, Mich.
Symposium on Microscopy, the sixth in a series sponsored T by the Armour Research Foundation of Illinois Institute of Technology, was held June 16 to 18 in Chicago. It covered asHIS
pects of electron microscopy, x-ray microscopy, organic qualitative microanalysis using the microscope. teaching of microscopy, and unsolved problems in the microscopy laboratory. There were no prepared talks, and the discussions were wholly spontaneous. The topic for the firpt session was: “Should the Light Microscopist Buy an Electron Microscope?” Jack Kelsch, Interchemical Co., served as panel chairman; panel members were F. Gordon Foster, Bell Telephone L.i’)oratories; A. G. Huckle, Imperial Paper and Color Corp.; Xian Kirkpatrick, American Cyanamid Corp. ; F F. Morehead, hmerican Viscose Corp. ; and C F. Tufts. Sylvania Produc’s Co. The arguments for the expected affirmative answer covered fairly well the general application of electron microscopy. Many advantages of a combination of light and electron microscopy were presented. C. F. Tufts noted the relationship between degree of resolution and cost and pointed out that whether or not cost is commensurate with the answers expected over the years depends upon a given company’s specific problems. Among the applications where electron microscopy is particularly adept are studies of micro structure and grain boundaries,
deformation, and etch pits, and determination of particle size. In all these applications, the light microscope is limited by low resolving power. In a discussion of the advantages of electron microscopy in the biological fields, Mary Rollins, Southern Regional Research Laboratory, pointed out that in a study on problems of high speed propulsion, the detection of a double, rather than single lining to walls of pulmonary arteries was possible only with the increased resolving power of electron microscopy. G. J. Socha, University of Wisconsin, reported on the use of electron microscopy by cytologists in studies of chromosome structure. Its use in virology is well known. Considerable attention was given to specific instances in which the so-called overlap region of light and electron microscopy is needed. J. J. Kelsch cited the study of the effect of particle size, shape, and efficiency of grinding on pigments, where important subtle differences are just beginning to be detectable as the limit of resolution of light microscopy is reached. The topic for the second session was x-ray microscopy. Serving as chairman was Sterling Newberry, General Electric Co., with panel members: dlbert Baez, University of Redlands; Jackson Clemmons, University of Wisconsin; Harold Sherwood, Eastman Kodalc Co.; and Thomas Turnbull, North American Phillips Co. Newberry presented a brief history of x-ray microscopy, and defined the present status of this rapidly developing field. Advantages include the large gain in resolving power due to the great decrease in wave length, the large depth of field, and the high penetrating power. Moreover, wave-length-dependent
1844
ANALYTICAL CHEMISTRY
absorption oharacteristics of matter are suoh that x-ray microscopy may he used for microchemical snalysis. Figures 1and 2 show the General Electric x-ray microscope; typical radiomicrographs are shown in Figures 3 and 4. Three met,hods of x-ray microscopy are now in use. The simplest, contact radiography, records microscopic information by the irradiation of an object in contact with the film. Magnifies, tion is entirely due to photoenlarging. The advantage is simplicity; limitations lie in the enlargement proces8. H. F . Sherwood reports increased definition by the use of a vacuum exposure holder, which ensures good contact between the specimen and the film emulsion. When double-emulsion film is u e d , such Kodak Type M, the back emuleion is either removed or restrained from development.
PHOTOGRAPHIC PLATE
++I
CONDENSER LENS
ELECTRON GUN-
-20 KV
proposed by Gabor, which may be termed microscopy by r e constructed waye fronts. An i m r t ~ n gsystem is devised such that diffraction fringes are plainly evident. The image is known as a hologram. Illuminating this rvith a source of radiation, and reveming it through an optical system, an image of the original object is reconstructed of the same size as the object.. However, by changing wave lengths, the reconstructed image will be larger by a ratio of the two wave lengths. Thus, employing x-rays initially, and reconstructing with visible light, a magnification of 5000 to 1 may be achieved. An important application of x-ray microscopy in chemical analysis was discussed b y B a a . The mass absorption coefficients of x-rays for an absorber of fixed atomic number follow a curve which has the general shape illustrated in Figure 5. The discontinuity in the mms absorption coefficient a function of wave length is associated with the binding energy of the electrons in their orbits. Considering a specific example, copper exhibits a discontinuity a t about 1.38 A. If a microscopic object containing copper is illuminated with monochromatic radiation of 1.30 A., the absorption will be high. It will be low with radiation of 1.5 A. If two pictures are mzde using each of these wave lengths, t h e copper in the sample will be dark in the first and light in the second. This is the qualitative aspect of the method; densitometry techniques permit its quantitation. Engstriim claims the detection of chemicals t o a concentration of about lo-” gram. Jackson Clemmons discussed an adaptation of the above method to biological materials. Such an adaptation, termed quantitative historadiography, involves the calcuhtion of mass absorption constants for thin tissue slices, using nitrocellulose as a reference standard. The concentration of water as well as organic matter may be determined on a given portion of a tissue section.
Figurell. Basic design of General Electric x-ray microsoope
The limitations of this method are overcome, t o some extent, by a second method, by which a shadow of an object very near a point source of x-mys iu projected onto a film a t some distance from the object. Magnification is achieved by virtue of divergence of the x-rays before the iniage is formed on the film, This minirniaes the limitation of film resolution, as little photaenlasging is necessary. Magnification is increesed as the distance from the murce to the object decreases, and the final resolving power is a function of the diameter of the source. A. V. B a a , reporting on his work at the University of Redlands, improves resolution by using tube de9igned by Cosslett and Nixan a t Cambridge, in which electrons are magnetically focused on a very thin metal target. The width of the electron beam a t convergence on the target is of the order of 1 micron. Thus, this is essentially the width of the “point” murce, provided the target metal is thin enough. A great advantage lies in the fact that the target is also the tube window, which permits close proximity of the object to the target a t atmospheric pressures. The ability to work a t normal pressures, thus obviating the problem of specimen dcsiccation, is particularly appealing to the biologist. Tho General Electric Laboratories have devised au x-ray microscope vhich uses electrostatic rather than magnetic focusing. A third method of x-ray microscopy utilizes focusing by reflection. Total reflection does occur, even with x-rays, a t angles approximating graaing incidence. Use is made of a system of curved “mirror” surfaces: A single pair of reflectors produces a line image from a point. A second pair of reflectors, whose effective surfaces remain perpendicular to those of the first, produces a point from this line image. Thus, divergent beams may be focused into convergence. At present, the resolving power of existing instruments of this type is estimated a t 0.5 micron. Considerable attention has been given the method originally
Figure 2.
General Elect mlerosoope
H. F. Sherwood announced the a,vailability of an extensive bibliography on microradiography and soft x-ray radiography fromtheMedicalDiviEion, EastmanKodak Co., Rochester4,N.Y. “Organic Micro-qual. Using the Microscope” mag the topic of the next session. D. E. Laskowski, Armour Research Foundation, was chairman; panel members were W. C. McCrone, Armour Research Foundstion; D. G. Grabar, Industrial Rayon Co. ; snd Ralph Johnson, University of Illinois.
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V O L U M E 27, NO. 1 1 , N O V E M B E R 1 9 5 5 McCrone emphasized convenience, the advantages of observing directly the results of tests, permitting ahigher degreeof confidence, as well BS greater insight as to what really happens, and the small sample required. An example cited was the applica, tion to the identification of chromatographic fractions, partioularly significant to the organic chemist. A more subtle hut powerful advantage was pointed out by Kirkpatrick: Microscopists have habit of retaining 3 v a t storehouse of mental images which are of great value in the solution of many difficult problems of identification or behavior. A consideration of separation and purification methods gave rise to discussions of (1) recrystallization on the microscope slide, whereby the best crystals, my for x-ray purposes, are "teased out" using a needle, (2) microsublimation, with the use of high vacuum for compounds that decompose a t elevated temperatures, (3) microdistillation, (4) mechanical separation on the microscope stage, to obtain samples for infrared, ultraviolet, and other analytical methods, and ( 5 ) thermal diffusion, Impurities in a melt will migrate to or away from the cooler portion of a microscope slide on a. Kofler stage (that section directly over the optical aperture). This was suggested by McCrone as the basis for a purification micromethod. Tufts suggested that eone recrystallation for purification of organic compounds might he successful if the thermal diffusion mechanisni can he utilized.
the addition compound, and the eutectic, if present, between the addition compound and the TNF. Should the unknown not form an addition oompound, aromatic derivatives of the unknown may often he made, which will then react to form an addition compound. Many of the classical methods of qualitative organic chemistry are readily adapted to micro levels, the reactions being carried out on a microscope slide. An exzmple is the 2,4dinitro- or p nitmphenylhydraaone formation as a test for the carbonyl group. As C. W. Mason, of Cornell, observed, the product of a lengthy reflux reaction very often may be quiokly obtained by simply melting the reactants together.
Figure 4. Aluminum-tin alloy (95 to 5 ) (X43) Taken with General Electric x-ray niicracope
Figure 3. Potato chip, showing salt crystals Taken with General Electtrio x-ray rnioroseopo t X 4 V
Only brief mention was given the methods of optical crystallography in organic qualitative analysis. Ralph Johnson discussed an electronic melting point device in which use is made of a hot stage with a very slaw rate of temperature increase. Light passes through the crystals on the stage through the microscope into a photaeleotric cell above the eyepiece. The variation in light transmittance as the crystals melt triggers an electronic device which records the temperature. In a discussion of classification reactions, considerable attention was giver, t,o mixed fusion techniques. The method is exemplified by the use of 2,4,7-trinitrofluorenone (TNF), which forms molecular zddition compounds with polynuclear aromatic compounds. Whether or not an unlcnown forms siuch an addition compound serves as a first classification; further classifications are made on the basis of melting point values for the unknown, the addition compound, the eutectic, if present, between the unknown and
Two half-day sessions were devoted to discussions of problems having an incomplete or unsatisfactory ansmr. Chairmen were Nick Galitsine, General Electric Co., and Charles Mareah, American Cyanamid Co. The combined panels included G. G. Cocks, Battelle Memorial Institute; John Facq, Toni Co.; F. B. Roseyear, Procter and Gamble; F. Gordon Foster, Bell Laboratorien; Fred Morehead, American Viscose Carp.; Oscar Richards, American Optical Co.; Mary Willard, Pennsylvania Stste University; and H. W. Zieler, W. H. Kessel Co. One of the first problems presented ims that of making stereomicrographs at high (400 to SOOX) magnification. Among solutions suggested was the half-aperture method, whereby a photograph is talien with h d f the condenser aperture covered; the other is taken with the other half covered. A slight loss of resolving power accompanies this procedure. Other suggestions were shifting of a decenterable iris diaphragm, and the use of crossed polaroids, eaoh covering only half the field of the condenser and with the two photomicrographs taken with the analyzer alternately parallel to each sector in the condenser. A problem of viscosity determination on 8 micro level was presented by McCrone. This specific problem required a determination involving little or no motion of a supercooled liquid melt. No promising suggestions were presented except perhaps magnetic movement of 1 t o 5 micron carbonyl iron particles. Difficulties in photographing moving objectfi, such as bacteria, or particles which exhibit Brownian movement, were mentioned by Cocks, who described a photoflash illuminator. I n cannection with the procedures of flash photomicrography wa6 a discussion of problem of light intensity and shape of light source,
ANALYTICAL CHEMISTRY
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the intensity becoming more significant in phase work. 0. K. Richards reported excellent results with his arrangement. McCrone mentioned the use of the cold stage in certain systems to “freeze” Brownian movement, permitting the use of standard exposures. The problem of sectioning fibers for electron microscopy received considerable attention. Mary Rollins suggested embedding the fibers in a 4 to 1 mixture of methyl methacrylate and ethyl methacrylate. Following sectioning and mounting on the grid, the methacrylate is dissolved out. The fiber may then be shadowed, and the electron micrograph taken. The advantages of pressure, rather than heat, in forming replicas for electron microscopy were mentioned. F. G. Foster pointed out the use, when microtomy is not applicable, of wax replication studied with the metallograph. This versatile instrument, though designed for the study of metal surfaces, may be applied to many other surfaces. In resinography, fillers and curing cracks are readily seen. Methods of surface preparation such as mild grinding and lapping are applicable, even on nonmetal surfaces. Dark-field and polarized light may also be used to advantage and are readily available with most metallographs. In connection with the measurement of sample thickness, disadvantages of the microtome setting method were mentioned.
Wave length
Figure 5.
1.38 A.
Mass absorption coefficient of coppei
.Among the methods presented were use of the micromanipulator to rotate a section on edge for measurement with the filar ocular, and the most common method, that of focusing from lower surface to upper with a calibrated fine focus. The instrumental, optical, and human errors involved were discussed. The interference microscope works very well for measurement of sample thickness when the refractive index of the sample is known. The question of versatility in instruments received considerable attention; opinions were divided as to the advantages of a “universal” microscope. The differences in opinion could be resolved, however, if the so-called universal microscope were not too badly cluttered with attachments and the price were low enough to permit several in each laboratory. The best universal microscope would seem to be a stand on which all possible accessories-i.e., interference, fluorescence, phase, polarized light, dark field, etc.-could be fitted as needed without disturbing the specimen. Other problems discussed involved projection of crystal images onto the slit of a spectrophotometer, for purposes of quantitating pleochroism, and methods of correcting for the various aberrations of lens systems. R. L. Seidenberg, Bausch & Lomb, discussed distortion, astigmatism, coma, and field curvature. Flatfield photomicrography may be achieved by introducing negative power into the lens system with a special eyepiece. This is an approach to the ideal, which could be surpassed, but appears to he optimum considering the cost of a more elaborate correction.
Another consideration is the sacrifice of resolution with gain in flatness of field, due to the decrease in the numerical aperature. The last session of the symposium was on the teaching of chemical microscopy. Chairman was F. Gordon Foster, Bell Laboratories. Panel members were C. W.Mason, Cornell University; Mary Willard, Pennsylvania State University; iilan Kirkpatrick, American Cyanamid Co.; and C. F. Tufts, Sylvania Products Co. A considerable portion of the discussions concerned the defining of features possessed in common by all good microscopists. These traits from the academic standpoint were outlined by Willard and Mason, and were general11 coincident with those from the industrialist’s viewpoint, as presented by Kirkpatrick and Tufts. A good microscopist is an “idea man,” well trained in the physical sciences. He is inquisitive, and as he is often the one who must bridge the gap between the other scientific fields, he is adaptable. The microscopy courses at Cornell University and Pennsylvania State University were outlined, and examples of industrial training programs were presented. C. W. Mason, whose teachings and writings have probably influenced the training of more chemical microscopists than those of any other man, summarized his feelings on the teaching of the subject. His emphasis was on supervised self-teaching, where one learns by doing. In teaching fiber identification, for instance, only an introduction should be presented. In ferreting out the rest of the information, the student develops his o m technique, and is caused to admit ignorance and ask questions. This latter characteristic continues to identify the best graduate microscopist. The importance of precise language Tvas also stressed: Poor communication is no less a barrier in teaching microscopy than in other fields. W. C. McCrone emphasized the need in industry for more microscopists, which reflects the need for more teaching of microscopy. The teaching plant must be adequate; many phenoniena must be shown, as well as described. As Mason stated, it is one thing to study phase diagrams-another actually to see that a eutectic is fine-grained. Most schools that teach microscopy merely integrate the n ork with other courses, such a$ instrumental analysis or mineralogy. Only a few, such as Cornell University, Pennsylvania State University, the University of Colorado, and Illinois Institute of Technology, give specific courses. EXHIBITS
Features of interest were exhibits of photomicrographs and various instruments available commercially. H. W. Zieler, W. H. Kessel Co., demonstrated a number of these instruments. Of interest was a Baker interference microscope with a lightshearing system, American Optical equipment for phase microscopy, and a Zeiss Winkel polarizing microscope with built-in illumination system, quintuple nosepiece viith individually centerable objectives, and an interchangeable binocular tube. A new Zeiss Winkel assembly for electron flash photomicrography included facilities for determining and setting the light intensity by means of a variable neutral density filter and a photoelectric measuring device. The Leitz Ortholux microscope with photomicrographic equipment and the Reichert research microscope, Zetopan, with features for visual observation as well as photomicrography, were demonstrated. Also shown were the Reichert heating stage and hot bar, both Kofler designed. Attached to a single upright was a photomicrographic unit n ith built-in light source (Orthophot) suitable for negative size 4 X 5 inches, as well as 35 mm., a time-lapse unit, in the lowprice range, with beam splitter, auxiliary lens system, and a Bolex 16-mm. movie camera, with range of exposure intervals from 1 to 1200 seconds. As a result of informal discussions, a committee was formed to plan future microscopy symposia with meetings in the East, perhaps alternating with Chicago meetings.
