NOVEMBER 15, 1936
ANALYTICAL EDITION
Chloropentammine-cobaltichloride was found to be the most suitable ammine for the precipitation of germanates. Partial analysis of the compound indicated its formula to be [CO(NH&C~]~H~G~(MO~O,)~. Since some of the results were satisfactory, a variety of conditions was tried in an attempt to learn what factor in the precipitation was responsible for the uncertain values often obtained. Experiments were performed in which (1) the quantity of molybdenum oxide was decreased to just a trifle over the theoretical amount and the acid concentration was reduced to a point a t which the ammine molybdate just stayed in solution, (2) the temperature of precipitation and the time of digestion a t this temperature were carefully controlled, (3) the temperature to which the solution was cooled before filtering was controlled, (4) the time of standing before filtration was varied, and (5) precipiwith shaking* None Of was made in a these variations led to ~onsistentresults; duplicate sometimes differed 10 to 50 per cent.
423
Literature Cited Baxter, Am. Chem. J.,28,298 (1902). (2) Baxter and Jones, J. Am. Chem. XOC., 32,303(1910). (3) Dakin, 2.anal. Chem., 39,789 (1900). (4) Ephraim, “Textbook of Inorganic Chemistry,” English ed. by P. C. L. Thorne, 2nd ed., p. 441,London, Gurney & Jackson, 1934. (5) Furman and Murray, J. Am. Chem. Xoc., 58, 1689 (1936). (6) Hillebrand and Lundell, Zbid., 42,2609 (1920). (7) Hynes, Malko, and Yanowski, IND.ENG.CHEM.,Anal. Ed., 8, 356 (1936). Ishibashi, Mem. COZZ.Xci. Kyoto Zmp. Univ., A12,23,39,49, 135 (1929). (9) Jorgensen, J . prakt. Chem., [Z] 18,243 (1878). (lo)Ibid., [2]23,227 (1881). Knowles, Bur. Standards J. Research, 9,1 (1932). Parks, G., and Prebluda, J. Am. Chem. SOL, 57, 1676 (1935). (13) Parks, M., Dissertation, Columbia University, 1930.
:ti;
RECEIVED July 31, 1936. Constructed from part of a dissertation submitted by Harold M. State in partial fulfillment of the requirements for the degree of doctor of philosophy, Princeton University, 1936.
Fluorescent Light Microscopy Possible New Applications to Industrial Research C. J. FROSCH’ AND E. A. HAUSER, Massachusetts Institute of Technology, Cambridge, Mass.
T
HE application of fluorescencethat is, visible light
emanated by a substance which is being irradiated with invisible ultraviolet light-in microscopy is not of recent date. However, the development has been slow, mainly because of the technical difficulty of obtaining sufficiently concentrated light of high intensity in the ultraviolet spectrum. Only comparatively recently has it been possible to devise such light sources. Haitinger’s (2) great contribution to the final improvement of the light source, as the result of a systematic spectrographic search, was the introduction of iron electrodes that give $n extremely efficient radiation between 3000 and 4000 A. These electrodes are held in specially constructed brackets, guaranteeing effective cooling. They are bored vertically, to enable filling this channel with a substance that prevents excessiveoxide formation, so that the arc can be easily started. This feature also assists materially in obtaining a very stable quiet arc. In collaboration with the firm C. Reichert, this new light source has been optically perfected, so that we have at our disposal today in the “Kam F” (Reichert catalog Kam, List 6094e) high-efficiency equipment for fluorescence microscopy which has already proved its value, notably in botany and biology. The voluminous literature concerned with microscopical ultraviolet studies in general fields, the textile and paper industries, dyestuffs, tanning agents, resins, rubber, pearls, botany, biology, and analytical chemistry, indicates the importance of such investigations, but, in the authors’ opinion, the ultraviolet microscope has not yet received the attention it so richly deserves, because its application to the study of industrial and theoretical physical phenomena has so far been neglected. The authors therefore decided to determine in a series of preliminary studies some possible uses of this instrument in industrial and theoretical physical research. The studies were carried out in a very general manner in order to determine as many applications as possible and the findings reported here are therefore only of a broad introductory nature. However, it is hoped that they may form the basis of further and more intensive research along the lines indicated, or sugI
Present address: Bell Telephone Laboratories. New York, N. Y.
gest other possible uses for the fluorescent light microscope that have not yet been considered.
EmulsScation The phenomenon of emulsification depends essentially upon the dispersion of one immiscible liquid in another. Consideration of the theories of emulsification and modern methods used in the preparation and study of these systems (1) led to the belief that visual observations of emulsification might be made with the fluorescent light microscope. The first essential in any study with the fluorescent light microscope is to obtain materials which are visible in the field of the instrument, because of the natural fluorescent characteristics of the components present or the addition of small amounts of adsorbable fluorescent substances, so-called “fluorochromes.” It is also most valuable in studying a system of two or more components to obtain as many fluorescent color differences in the excited visible light as there are constituents in the system, unless these can be clearly identified by structural or other properties. Since a twocomponent system is the simplest type of emulsion possible, it was deemed advisable to begin with the study of an emulsion of water and a neutral oil. Pure water is known to be nonfluorescing, so that it remained to select an oil which would eyhibit suitable fluorescence. A purified paraffi oil exhibits a very faint blue fluorescence which was not considered sufficiently pronounced for the studies in mind. There are other oils which exhibit very pronounced fluorescence, but when an inactive hydrocarbon, such as anthracene, is dissolved in paraffin oil it becomes very strongly fluorescent in the deep blue. When a drop of water is brought in contact with a drop of anthracene-treated paraffin oil on a u. v. transmitting slide and the system is immediately observed with the ultraviolet light microscope, using transmitted light, only the drop of blue fluorescing oil is visible in the field. [The magnification throughout these studies was maintained a t 99 diameters (11x objective, 9 x ocular) since this was found to give the best resolution of the phases in the systems considered.] As the time of observation increases, tiny crystals exhibiting
424
a light greenish blue fluorescence are seen to form in the oil drop close to the interface. The crystals, in a fairly wide band extending from the oil-water interface into the main body of the oil drop, are in constant directional motion almost parallel to the interface but gradually moving to the oil side of the oil-water interface, where they aggregate in clusters with continued but noticeably slower motion along the i n t e r f a c e (Figure 1). No motion or orientation of the crystals is n o t i c e a b l e i n the reHe0 mainder of the oil drop. Still another interesting effect can be seen along AIR the oil side of the oilinterface extend+ ANTHRACENE CRYSTALS water ing for a comparativelv FIGURE 1 shirt distance- from thk oil-water-air interface, where not only is no crystallization visible but also no tendency for crystal movement into these regions. The above admittedly preliminary experiment indicates visually the movement of crystals in an oil-water interface and no doubt analogous effects take place when solids are used in emulsification processes. The crystals might thus be compared to solid emulsifiers and it might be inferred from these observations that the stabilization of an emulsion with a solid depends upon the formation of a protective film of the solid at the interface in the phase which wets the solid most readily. The latter suggestion was not investigated in detail in the present survey, but it is felt that a complete investigation of emulsification with solids offers a very fertile field of research with the fluorescent microscope. It has long been known that ultraviolet light promotes crystal growth from solution, although the possibilities of this phenomenon have not as yet been sufficiently considered. The anthracene crystallization from the paraffin oil evidently took place from a solution which was not supersaturated with anthracene, since it was ascertained that this crystallization takes place in the absence of water, and continued irradiation of an oil drop with ultraviolet markedly decreases the intensity of the original blue fluorescence of the oil solution. This crystallization effect would indicate that the solubility of anthracene in paraffin oil is decidedly less in ultraviolet than in visible light. However, it has been shown (6)that anthracene is converted into dianthracene, a less soluble form, when irradiated with ultraviolet, the amount of the conversion being proportional to the amount of energy absorbed. This conversion effect is true for other organic compounds, but i t is also stated (6) that dianthracene is not fluorescent, whereas the crystals referred to above exhibited a decided greenish blue fluorescence. This would indicate either that these crystals are not dianthracene, or that anthracene is adsorbed on the dianthracene crystals, causing them to fluoresce, or contrary to the former statement, that dianthracene is naturally fluorescent. Further study is necessary to decide the issue, but it would seem that ultraviolet light might be advantageously applied in crystal-purification methods in spite of the possible formation of polymers, since these are generally reversible and are usually easily reconverted to the original compound. A solution of rosin in an asphalt-base crude oil fluoresces a greenish blue in the ultraviolet and remains stable with time. If a drop of water is brought in contact with a drop of this oil and the system is observed with transmitted ultraviolet light in the microscope, no change is noticeable a t the oil-water interface. Upon stirring a small amount of oil into the water drop in the field of the instrument, greenish blue
-
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INDUSTRIAL AND ENGINEERING CHEMISTRY
droplets of oil are seen to be dispersed in the water and it is particularly noticeable that these droplets are homogeneous in color structure. When a drop of 1 per cent solution of sodium hydroxide instead of water is brought in contact with a drop of oil containing rosin, a beautiful blue fluorescent band is visible a t the interface between the two liquids. This band is no doubt due to the formation of sodium resinate, which being capillary-active is adsorbed a t the interface, exhibiting its characteristic blue fluorescence. The effect of this adsorption is more strikingly demonstrated when the two drops are stirred together, for each droplet is observed to be surrounded with a strongly fluorescent blue ring (Figure 2). It should be pointed out that the visibility of this band is dependent upon the size of the droplets, because it is evident that this adsorption effect must take place around .the entire drop and is visible as a ring only because of the thickness of the droplets. This is readily demonstrated with incident light, for the oil droplets in pure water remain as before a greenish blue, whereas those in sodium hydroxide solution exhibit a deep blue fluorescent color throughout. It is generally known that sodium resinate tends to form oil-in-water emulsions, whereas calcium resinate tends to form water-in-oil emulsions. This suggested the study of an emulsion of the water-in-oil type by @ using calcium chloride instead of the sodium hydroxide used in the previous experiments. V////~-GREENISH-BLUE A drop of a 1 per cent solution of calcium chloride is brought BRIGHT BLUE in contact with a drop of rosinFIGURE2 treated oil and the system is observed in the ultraviolet microscope. A beautiful band of blue fluorescence is again noticeable a t the oil-water interface. When the two drops are mixed in the field of the instrument, a rather unstable dispersion is formed, but in this case the blue rings are not visible around the dispersed phase. This is to be expected, since the continuous phase is now oil which fluoresces a bluish green and hence obscures the blue color of the calcium resinate adsorbed on the surface of the water droplets.
,I . " , "
a
--
W&,
- BLUE
FIGURE 3
This study with the two types of emulsions suggested the possibility of using the fluorescent microscope to determine the dispersing phase in an emulsion. When a crystal of anthracene, which is oil-soluble, is added t o the above emulsion prepared with calcium chloride, a blue fluorescent halo develops around the crystal and gradually spreads through the system. This demonstrates that the emulsion prepared with calcium chloride is of the water-in-oil type. If the same procedure is now repeated with the sodium hydroxide emulsion as used above, no halo is formed around the anthracene crystal, indicating that the dispersing phase in this case is water and the emulsion is of the oil-in-water type (Figure 3)-
Ore Flotation The concentration of ores by flotation depends essentially upon the preferential surface conditioning of minerals to cause
NOVEMBER 15, 1936
ANALYTICAL EDITION
them to become attached to air bubbles and thus be floated, whereas the accompanying gangue or tailing, being unaffected by the treatment, remains in equilibrium with the water phase and hence sinks. The principles of ore flotation as they have been suggested by both practical application and theoretical research have been adequately considered in the literature ( 5 ) ,but the nature of the mechanism of this action is for the most part still very indefinite. Actually following the process of ore flotation by some visual method would therefore seem extremely important. This suggests the use of the fluorescent light microscope if one could distinguish differential fluorescence in the systems studied. The particular system considered in this article, the flotation of galena from sand with oleic acid and amyl alcohol, is only one of a great many possible systems, but it demonstrates the value of the ultraviolet light microscope in such studies. When a mixture of galena, sand, amyl alcohol, oleic acid, and water is shaken in a test tube and the tube is held in the path of ultraviolet light, it is immediately evident that the foam on the surface is very strongly fluorescent in blue, while the main body of the liquid is much less so. This effect is more marked a t fairly low concentrations of oleic acid and amyl alcohol if the proportions of the solid constituents in the system are kept relatively constant. Since the foam, according to suggested theories, contains the galena ore as well as the greater portion of the oleic acid and amyl alcohol, a study of the ore-flotation process with the fluorescent light microscope seems feasible. I n actual ore-flotation processes the efficiency of the ore separation is dependent in part upon the size of the solid particles in the system. A few tentative experiments were therefore made to determine the most suitable particle size for the studies in mind. Solid particles of sand and galena passing through a 20-mesh but retained by a 48-mesh screen seemed to be best. Galena ore observed in the fluorescent light microscope does not fluoresce with transmitted ultraviolet light, although a very faint reddish fluorescence is noticeable with incident light, and the wetting of the galena particles with water produces no visible alteration in the ultraviolet. When particles of galena are shaken with water having a drop of oleic acid floating on its surface, the excess solution is decanted off, and the wetted particles are observed in the fluorescent light microscope with transmitted ultraviolet light, all the galena particles are seen to be surrounded with a definite layer of greenish blue fluorescent material, characteristic of oleic acid, whereas with incident light only homogeneous greenish blue particles are visible. It is obvious that the whole surface of the galena particles must be coated with oleic acid, obscuring the particle with incident light but indicating it as a black body with transmitted light. Transmitted light, therefore, serves to indicate a cross section of the adsorbed materials surrounding the mineral, and being therefore much better suited to the observation of the details t o be studied was used through the remainder of this study. When a drop of amyl alcohol is brought in contact with the above wet agglomerate of galena while the layer of oleic acid and the dark wall of one of the galena particles are in constant observation, an explosive reaction occurs-i. e.; the layer of oleic acid is rapidly made more diffuse and tiny galena particles are observed to have broken off the wall of the galena crystal. The latter phenomenon is thought to be due to cracks or capillaries in the surface structure of the ore, and the spreading of the oleic acid by the amyl alcohol is so violent and rapid in action that the outside edges of the mineral are shattered by the forces exerted in these hypothetical crevices. There is also the possibility that galena may be made up of a plate or disk-like lattice or units analogous to the structure of “Dickite,” a kaolinite clay mined in Colorado.
425
If galena particles are first shaken with water and amyl alcohol and the excess solution is decanted, the particles are observed to be surrounded with a very diffuse halo of blue fluorescence, the intensity of which is dependent upon the concentration of amyl alcohol originally used. When a drop of oleic acid is brought in contact with this wet mass, observation of the edge and surrounding of one of the galena particles shows that the drop passes to the surface of the mineral as a diffuse layer without any bursting of the galena particles. This is shown not only by the actual motion of the oleic acid but also by the decided increase in fluorescence surrounding the particles and a change to a greenish blue. When sand is observed in the ultraviolet, only a very few particles are noticeably fluorescent in the yellow and red, probably because of some impurity, and no visible alteration of the particles occurs with the addition of water. If a few particles of sand are shaken in a test tube containing water and oleic acid and these wetted particles are observed in the fluorescent light microscope, very few of them exhibit the greenish blue fluorescence of oleic acid around their edges, and in the cases where this is true the bands of fluorescence do not always surround the mineral but are in some cases confined to only one edge or spots. The latter effect is probably due to the presence of mineral impurities preferentially wettable by oleic acid in the sand particles which might conceivably be present in only localized areas of the sand. A drop of amyl alcohol added t o this agglomerated mass causes some agitation and pushing apart of the sand particles, while the blue fluorescence of the alcohol becomes evident throughout the system. These experiments in the study of ore flotation with the fluorescent light microscope have so far considered each solid component in a separate system. I t now remains to study sand and galena in a single system. When galena, sand, water, and oleic acid are shaken together and the wetted solids are observed with the f l u o r e s c e n t microscope, it is noted that many particles are coated with a laver exhibiting a greenish blue”fluorescence, whereas the -GALENA PARTICLE remaining particles do not show this effect, The par- M -GREEN-IXUE (OLEIC ACID) ticles exhibiting the band of SAND fluorescence are readily identified as g a l e n a b e c a u s e of FIGURE 4 their c h a r a c t e r i s t i c cubical shape (Figure 4). The addition of amyl alcohol to the system has the same explosive effect on the galena as previously described. Sodium n-amyl xanthate, a collecting agent commonly used in ore-flotation practice, when used in conjunction with oleic acid does not differ appreciably from amyl alcohol in its effect and therefore needs no further discussion here.
’*-
General Studies The experiments discussed above have been confined to specific practical problems and their study with the fluorescent light microscope, but during these experiments certain other possible uses of the fluorescent light microscope were suggested. One of the most difficult problems in the study of wood structure by microscopical methods is the preparation of microtome sections for use with transmitted light. This is especially true in a study of wood in a partially destroyed state, such as that resulting from advanced decay. The Leitz Company was the first to offer a solution for this problem
426
INDUSTRIAL AND ENGINEERING CHEMISTRY
by introducing the “Ultropak,” which does not require thin sectioning since it employs incident light. As the studies considered here are not concerned with visible light, the discussion will be limited to observations with the fluorescent light microscope. When a block of poplar wood is observed under the fluorescent light microscope with incident light, which does not require microtome sections, it exhibits the characteristic lightblue fluorescence common to wood. Although the structural details of the wood are extremely clear, no differential fluorescence in the structure is indicated. Koehler’s (4) suggestion of the use of “colorless dyes” in fluorescent light studies prompted a study of their use in determining the different structural components known to be present in wood. When sections of poplar are submerged in various fluorescent dyes and are then observed under the u l t r a v i o l e t microscope in their wet state, only the primulin dye (a yellow cotton dye o b t a i n e d by heating paratoluidine with sulfur and then sulfonating the reaction product) shows -AREA PENETRATED preferential adsorption AS SHOWN BY on the wood section. FLUORESCENCE OF This is e v i d e n c e d by SOLUT’oN distinct differential color FIGURE5 f l u o r e s c e n c e in pink, blue, and green. These fluorescent colors are not dispersed on the wood in a heterogeneous manner, but are homogeneously dispersed on definite structural elements of the wood. The selectivity of the primulin is not altered with drying the wood section. In the course of the above studies with wet wood sections, the clearness of the liquid menisci in various pores was particularly noticeable. This suggested the possibility of studying the rate of penetration of liquids into these surface pores. A drop of anthracene-treated oil was brought in contact with the outer surface of a wood block and the movement of the menisci observed under the fluorescent light microscope. Most of the menisci moved through the field rapidly, only a few moving slowly enough to be readily followed. A complete study would therefore require the selection of a suitably viscous oil or fluid, In any case the anthracene-treated oil upon filling the pores produced a beautiful picture of the capillary structure of the wood surface, for the pores fluoresced bright blue whereas the capillary walls remained greenish blue. One of the most difficult problems in the study of commercial impregnation of porous materials with liquids is the determination of the depth of penetration when the penetrating substances are colorless or their color is obscured by the solid, The fluorescent light microscope offers a solution to this problem, for the depth of penetration is shown either by the natural fluorescence of the penetrating liquids themselves or by that of small amounts of fluorochromes added to the liquids before impregnation, The latter is better than the use of colored dyes, because visible color is undesirable in most finished products where colorless liquids have been used for impregnation. The use of the fluorescent light microscope for determining the depth of penetration has already found an application in the leather industry (determination of depth of tanning) and it is hoped that i t will prove valuable in other industries concerned with impregnation processes (Figure 5). Another possible use is in the identification of molds and fungi, of extreme importance in many industrial and scientific fields. This may be based on either natural or incited differences in fluorescence among the organisms. A few wooddestroying fungi were observed with ultraviolet light and
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exhibited natural greenish blue fluorescence in all cases except one, where pinkish fluorescence was noticeable. A number of other uses of the fluorescent microscope have come to mind through this series of studies, but it seems rather futile to attempt a discussion of them without first tentatively verifying their possibilities. Although some of the suggested applications may prove of little value, the authors are confident that others will be most valuable in broadening our knowledge of the mechanism of processes to be investigated. In publishing this admittedly very superficial general survey a t this time the authors hope to incite others to make increasing use of this new experimental tool in research activities that have heretofore evaded visual observation. Theoretical details and the application of fluorescent light microscopy in the study of adsorption and other surface phenomena are presented in another article (3). Summary Since the construction of highly efficient and highly concentrated ultraviolet light sources, fluorescent light microscopy has proved of constantly increasing interest and value in a great number of applications-for example, in biological and botanical research, in inorganic analytical chemistry, as a routine control method in the textile and paper industries, etc. However, fluorescent light microscopy lends itself perfectly to the study of typical colloid physical or chemical processes or reactions, and permits for the first time visually demonstrating and following such processes as the production and destruction of emulsions, and the influence of different types of emulsifying agents on the stability of the system. It offers a simple, quick, and reliable method of determining types of emulsions which so far have been difficult to ascertain. Fluorescent light microscopy promises to become a valuable new method in detailed studies and routine control of flotation processes. It is applicable in studying the degree of penetration into porous absorbent matter-for example, in the tanning of leather with different types of natural or synthetic tanning agents. This new technic is of value in the study of wood preservation and in the detection of molds or fungi. Since modern fluorescent light microscopy permits the use of incident as well as transmitted light, it becomes unnecessary to prepare microtome sections and to apply selective dyeing methods. The use of “colorless” dyes of high fluorescing power further broadens the applicability of this method in cases where the substance itself does not emanate fluorescent light if radiated with invisible ultraviolet light. Literature Cited (1) Clayton, W., “The Theory of Emulsions and Their Technical Treatment,” 3rd ed., Philadelphia, P. Blakiston’s Son & Co., 1935. (2) Haitinger, M., ilfikrochemie, 9, 430 (1931). (3) Hauser and Frosch, J. Optical SOC.Am., in press. (4) Koehler, A., 2. wiss. Milcroskop., 21, 129, 273 (1904). ( 5 ) Petersen, W., “Sohwimmaufbereitung,” Dresden, Theodor Steinkopff, 1936. (6) Spielmann, “The Constituents of Coal Tar,” p. 94, London, Longmans, Green & Co., 1924. REC~IVE September D 18, 1936.
