436
A N A L Y T I C A L CHEMISTRY
visual portion become transparent w h e n radiatedwith infrared (9). Microscope optics are transparent to near infrared as far as 2 fi. The sensitivity of the eye falls off "t about 7000 A. Ordinary infraredsensitive photographic mateFigure 8. Molyhdenite rials have their Interference figure shorn with 4-mm. 0.85 Deak sensitivitv N.A. obj,j,tire, is uniaxial &tabout 8000 The infrared image tube (1P25,14) used in World W&: I1 in the Sniperscope (11) has a peak sensitivity rtt about 8200A.
A".
An infrared image is formed on a photocathode, which gives off electrons proportional to the infrared radiation it receives. These electrons are accelerated in an increasing potential field
and are brought to focus on a DhosDhor screen a t the other end ~
~
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over the evepiece of a convent:okl ~&croscme. The source of light used &this work is a carbon arc lamp fitked with a Corning No. 2540 filter to absorb the visual light and transmit the infrared portion of the spectrum. An example of this use of infrared (Fimre 8) shows the uniaxial interference fieure of rnolvh~"~ denite, mineral opaque in visual light.
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LITERATURE CITED
(11 Biickatrbm, H. L. T.. Arkiv K m i M i n e ~ a lGeol.. . 13A (193'31 (2) Bailley, Ren~,Bull.acad.roy.Belg., l2,791-822(1938). ' (3) Brumberg. E.M..Bull. Aead. Sci. (U.S.S.R.), 6 , 3 2 4 0 (1942). (41 ( L n n . d m . 1947.41-0..~ " SOC. . . Burch. C. R.., Proe. Phvs. ( 5 ) Foster; L. V.. clnd Thiel, E. M., J . Optical SOC.Am., 38, 689-92 ~
~~~
i l O~ d P ," ~ " , . ~
Johnson. B. K., Pmc. Phgs. Soc. (London). 1939, 1034-9. Kasha, M., J . Optical SOC.Am., 38, 929-34 (1948). Koehler. August,Z. wiss. Mikroskop., 21, 129-65 (1904). Zbid.. DD. 273-304.
. L. ~, V., J . Optical SOC.Am., 21, ~
Trivelli, A. P. H., and
Foster.
,?A ,lo?,, .I_~_"__,_ (14) Zworykin, V. K., andMorton, G.A.,Zbid.,26,181 (1'336).
15, 1948. R ~ a ; ~ v eNovember o
Rpplication of Fusion Methods in Chemical Microscopy W. C . MCCRONE, Arrnour Research Foundation, Chieogo, 111. oal and research applications of old but little used technique (fusion Is) are emphasized. Many applications are possible in the fields of and phase rule research, as well as qualitative and quantitative analysis.
F.
memaas represent a group of techniques which the uucroscoplst . oan use in investigations of organic compounds, or more generally fusible compounds. This tool can be used for characterization and identification of fusible compounds, determination of phase diagrams, punty determinations, qualitative analysis, q w t i t a t i v e analysis of binary and even polycomponent systems, study of mechanism of crystal growth, and study of other changes in the solid state such as rcerydtrtlliarttion, grain growth, ttnd boundary migration. The techniques themselves include all observations made during heating of a few milligram of the material on a microscope slide, during solidification of the melt, during cooling of the preparation, as well as observations on the erystalliaed material a t room temperature. In addition, data obtained from observation of a mixed fusion with a reference substance may also be included. The hasis for the application of fusion methods was laid by Lehmann ( 4 )in 1891, when he pointed out that crystallization of a n organic compound from the melt is very characteristic. He listed a qumber of properties that can he determined on crystals from the melt, and pointed out the value of fusion methods in studying phase diagrams. Identification of fusible compounds by fusion methods permits a very rapid analysis with small quantities of material and requires relatively little specialized training or equipment. Complete op1 UD~ULY
tical crystallographic description of a given compound may require from a few hours to several days, whereas complete fusion analysis of a new compound seldom requires more than an hodr. Furthermore, a n unknown compound in a limited category-cg., organic acids or vitamins-can usually be identified in less than 5
Figure 1. Sublimate of Hexamethylenetetramine on Microsoope Slide. 50X
V O L U M E 21, NO. 4, A P R I L 1 9 4 9
437
perature necessary in phase diagram work or charactorisation and identification a i organic compounds can he made using this hot stage. Molecular weights can be detormined b y the Rast mieramethod, using camphor in small flattened capillaries or other less volatile solvents in open cover glass preparations. Another similar hat stage applicable for study of fusion behavior is described by Mittthews (6). TECHNIQUES
Tho use of fusion mcthods requires observation of hot preparations on the microscope stage. It is necosssry, therefore, to ohserve certain precautions in order to avoid damage to the microscope.
Figure 2. Crystals of 1-Aminobenzothiazole Crystallized from Melt. 2SX
I n general, low magnification (20 t o 50X) with crossed Nicols is most useful. A 32- or 40-mm. objective is essential, for an ohjec-
Gse spaces result from deoompoaition from melt
Figure 3.
Growing Crystal Front from Melt. SOX
Upper. Tefranitroanilioe Louer. A o e n l sslievlio acid.
Crossed Nioola
minutes, if the fusion data for that compound have once been determined. So many characteristic properties can be observed on crystals from fusion that one or two easily recognized and typical properties can be remembered for each compound; hence a complete check of all properties is not always neoessary. A hot stage is not necessary for many of the techniques, although it is very useful for accurate determination of melting points. Kofler and Kofler (3) have developed a carefully designed microscope hot stage (available through Arthur H. Thomas Company, Philadelphia, Pa.) which when carefully standardized gives both x c u m t e determination and close control of temperature on the stage. This is done by careful design and by carefully calibrating the thermometer in the hot stage with compounds of known meltingpoints. Very accurate determinations of the tem-
Figure 4.
Polymorphio Transformation
Upper. Cnsfallins film of vanillin showing eolid-solid polymorphic fransformnfion. Dark s-as shown growing as new c n s t a l s of stable phase Center. Choleateryl acetate, showing spheritea of rmm femp r n t u r s stable modifioation growing at expense of dark Liquid crystalline form I n w s r . Mercurioiodide. Red form, dark. Yellow form, light
ANALYTICAL CHEMISTRY
438
Figure 5.
nation may show melting and subsequent crystallization from the melt because the time of heating is so short by this means that extensive decomposition cannot take place. During coaling of the preparation additional properties may be determined. Tendency to Form a Supercooled Melt. Many compoundse.g., thymol-characteristically supercool and must be seeded in order to induce crystallisrttian. Most compounds, however, resist all efforts to induce them to SUpCrOOOl very far helow the melting point. Another group of compounds--e.g., n-butylamine hydrochloridesolidifies to a glass unless the temperature is maintained just below the melting point, so that crystallization can occur. Rate of Crystallization. Here again there is considerable variation in the rate at which different organic compounds crystallize so slowly from the melt. Som-.g., o,p'-DDT-crystallise that several hours would be required for complete crystallization of a fusion preparation, while others, especially when supercoolod-.p.. rtcettmilide-ervst,nli~~aomnlet,elv in a frn.ct,ion of a second.
Crystalline Film of p-Dichlorobenzene. lOOX
Twin banda reaulfing from msohanioal
PT~S~UV0 C"
wver d s s a
Figure 7. ~~
Interference Figure of Trinitrotoluene ation (0.85 N.A.)
Form of Crystal Front (Figure 3). Crystallization from the melt usually proceeds in a typical manner for each compound. The crystal front will often show reproducible profile anxles.
for this purpose iscdled a n&ed fusion and is described herewi'th.
Figure 6.
.
Shrinkage Cracks in Tetraohlorohydroquinone. 25X
.. ..
I
"
% i
2
-
F i g u r e 8.
Mired Fusion
Upper left. Component A melted and allowed to crystallias Upper right. Component B melted and allowed to run under cover glnsa and oryetallire Lower left. A11 of B and part of A remelted to form sone of miring Lower right. A and B recryatallimd to sone of mixing
V O L U M E 21, NO. 4, A P R I L 1 9 4 9 A number of other properties in addition to profile angles can be determined on the crystal front. A more or less complete list, in addition t o those listed shove, is as follows: polymorphism (Figure 4), refractive indexcs relative t o the melt, birefringence, dispersion, sign of elongation, twinning (Figure 5 ) , anomalous polarization colors, shrinkage cracks (Figure 6), gas bubbles, and correlation of crystal optics with crystal geometry (Figure 7). The Xoflerj (S) use as further characteriaation the eutectic melting point of each compound with several standard compounds. This gives several numerical properties which are very distinctive and which are tsbulated for analytical purposes. One t.housand eomnounds have been analyzed by these means and tabulated (5). A mixod fusion can be used as suggested above to give further
439
information for characterization purposes or to determine the phase diagram in a two-component systom.
A mixed fusion (Figure 8) is carried out in three steps. One t o 2 mg. of the higher melting component are melted under the cover glass and allowed to solidify; the low melting component is then placed a t the edge of the cover glass and heated so that it melts and runs under the cover glass into oontact with the high melting component; next, the preparation is reheated so that all of the low melting and some of the high melting component melts. On cooling, crystals of the high melting component will grow up t o the zone of mixing and stop. The low melting component will then crystallize from its side of the zone of mining and stop at.the zone of mixing. APPLICATTONS
+..,"
.c. i L n y v v y L
...
nn,mnmnnn+. in the zone of mixing Can The behavior be used for a number of purposes. Far example, if the low melting component is, or remains, a liquid-ex., benzyl alcohol, nitrahenzeno, a r d o r , or thymolL-orystals of the high melting component will erow into the Bone of mixina, dimst, and develop into well.~ formed crystals (Figure 9). These can be used for careful study of tho orvstsl -geometry and correlation of the geometrical and optical properties. The mixed fusion can also he used as an identity test; if the two components treated in this manner are identical, there will be no zone of mixing and crystals from one side will grow complotely ~tcrossthe preparation with no discontinuity in rate of growth a t the zone of mixing. On the other hand, if the two components are different there will be a discontinuity in rate at the zone of mixing. Furthermore, in general, a t least one additional phase will q p e z r , indicating either addition compound or eutectic formation. This would be a certain indication that the two samples are different. Systems showing solid solution formation will show a discontinuity only in rate of growth a t tho zone of mining; this is, ~y.A.yy..b
y.
~
Figure 9. Well-Formed Crystals of Pioric Acid Formed b y Mixed Fusion w i t h Thymol. 50X
Figure 10. Sequence Showing Mixed Fusion between Coumarin (Left) and Vanillin (Right), I l l u s t r a t i n g Eutectic Formation. 50X
ANALYTICAL CHEMISTRY
440
Figure 11.
Sequence Showing Mixed Fusion of T r i n i t r o t o l u e n e a n d Mononitronaphthalene, Addition Compound Formation. 50X
hoaevcr, sufficient to show that the two components are not identical. Finally, because the zone of mixing contains the complete composition gradient for the two-component system, the phases that appear on crystallieation will be characteristic of the phase diagram for that system. The general type of phase diagram for any two fusible compounds can, therefore, he very quickly determined. The simplest phase diagram is the somewhat trivial case where the two components are completely insoluble in each other. There are a number of important examples of this type, some of commercial importance-e.g., Amatol; T N T with ammonium nitrate. The usual types of phase diagram are, of course, eutectic systems, addition compound systems, and solid solution systems. Each can he readily distinguished through use of fusion methods, as shown in Figures 10 and 11. It is possible, using a carefully designed hot stage, to determine melting points or transition temperatures and by making up known mixtures of the two components to determine the compositions of addition compounds or eutectics. In this way the entire phase diagram can be accurately determined microscopically. The phase diagram in a given two-component system will be different for each of the polymorphic forms which might be present. If, for example, each of the two components could exist in two different polymorphic forms there would he, not a single two-component phase diagram for this system, but four separate and d i e tinct phase diagrams depending on the polymorphic forms involved. The most complete cavemge to date of the application of fusion methods to determination of phase diagrams can he found in book recently published by Kofler and Kofler (S). It is often desirahle to obtain a quick answer to the questions: Is this compound pure? About how much impurity is present? Which of two samples of a given compound is purer? These questions can be quickly answered by observing the crystal front as it grows (Figure 12) and by examining the crystalline film from
Illustrating
l
fusion for eutectic melt. In a few cases the impurity is isomorphous, hut in all other cases a residual melt will he apparent after sofidificationof the principal component. The presence of residual melt answers the first of the above questions; the amount of melt or relative amount of melt answers the second and third questions. This is a very sensitive test for impurities and should be used on all samples intended for micrortnalylysis. It may he used sa well on organic compounds to follow purification by recrystallization, ete. Fusion methods can also be used in various ways for the quantitative analysis of mixtures. The properties of a, mixture that can be used to determine the percentage composition of a mixture include: rate of crystd growth of the major component (5), refractive index of s, binary melt (S), habit of crystals from the melt
V O L U M E 21, NO. 4, A P R I L 1 9 4 9
441
the rejection of gas huhhles are a few of these manifestations. Having observed orystallization of organic compounds with a microscope, it is difficult, if not impossible, to forget the appearance of the crystal front when observing crystallization on a larger scale or observing materials that have crystallized from the melt. Many of the properties and much ai the behavior of such crystalline phases can be more readily understood and interpreted on the hasis of their appearance on a microscopic scale. Anothor very important field for iuvcstigation by fusion methods is boundary migration. This was first observed while watching the crystdlisation of organic compounds from t h e melt and is apparently closely related to g a i n growth and recrystallization in metals. It can be described for organic eompounds somewhat as follows: Figure 13. Crystals of p,p'-DDT Showing Secondary Feathery G r o w t h Due to Boundary Migration a t Boundaries of P r i m a r y Spherites. IOX (71, optical and geometrical properties of mixed crystals (11, and melting point composition diagram. Each method is restricted to a definite class of cornpound and to definite conditions of analysis-for example, the method based on optical propcrfies can be used only when the two components form solid solutions. In addition, this method 8.6 m ell as the method based on refractive index of the melt can he used only in systems of two components, whereas the method based on crystalliestion rate can he used on polyeomponent systems but only under special conditions to determine the amount of the major constituent. Mitchell (7) has recently shown that the crystal habit of adipic acid from the melt vanes considerably hut reproducibly on the addition of small amounts of succinic acid. This has been used to indicate the amounts of succinic acid in samples of adipic acid. The use of FefraCtive index of the melt as m analytical method has been developed by the Koflers (8). The technique involves use of a series of glass powders (available through Arthur H. Thomas Company, Philadelphia, Pa.) of accurately known refractive index. Known mixtures of the two components are then prepared, mixed, and melted with the glass powders until that powder having an index lower than the melt is obtained. On further heatinr wit,b this elass powder a temperature is reached a t which the two refractive ind exes become equal. A curve is then prepared of temperature verrins composition for each glass powder required t o cover the composition range. A few of the classicd methods of microscopicd analysis can also be used to analyze binary Inixtures of organic compounds and one, areal analysis, is of value in analyzing systems where the components are insoluble in both the liquid and solid states. In this case the relative areas of the two components in a thin cryshlline film are proportional to the relative percentages of these components. The ease with which thin crystalline films em he studied microscopicdly makes i t possible to study the kinetics of crystal growth. In particular, the properties which affect the rate a t which molecules become orlented on and enter into a crystal front, can he studied by studlying the effectof variations in molecular shape, size, polarity, etct, on rate of crystdlization. I t has been found by this means tha t the rate of crystallization of a given compound in a binary mixt ure may he more rapid than in the pure state (#). The compomId, p,p'-DDT, for example, will grow almost 100 times as rapidly in mixtures containing 20% Cellosolve as in the pure state a t the same temperature. Other manifnn.+s+inna nf t h o manna? .l.y in 1.vhich crystallization occurs can also be studied by this means, It is possible to observe in the crystdlilliastion of organic compounds from the melt many types of behavior which must occur on a larger scale in industrial processes from the casting of explosives to the casting of metals. Stresses built up during er,y:ystdliastion,shrinkage cracks that result, and ~~~
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~~
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~
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relative-to each other. 'Some organ% compo&ds under these conditions will show a growth of one crystd into and through a second crystal of different orientation. In all cases the relative orientations are the mme for a given direction of growth and a given system-for example, p,p'-DDT crystallines as spherites, composed of needlelike crystals growing radially from each center. The spherites will grow until they contact one another and the intersection linc of each spherite will form a contact area for needle crystals from two adjoining spherites. The angle between needle crystals on opposite sides of the contact zone will vary from almost 0" t o 180". When the angle is 90" the needle crystals will show a strong tendency to grow into and through each other, so that a secondary growth of the same phase is formed (Figure 13). The tendency for this to occur will decrease as the angle between opposing crystals approaches either 180" or 0". This behavior has been ohserved for only a few organic compounds-e.g., p,p'-DDT, vitamin K, and 8-oxyquinoline. It Seema probable that this may be related to another type of grain growth shown by other organic systems such as ohloropropane wax, camphor, and carbon tetrabromide (8).
It has been shown conclusively that when boundary migration occurs the secondary crystals are identical with the original crystals and that the orientation is the principal factor determining the direction of migration. Further study of organic compounds using fusion methods should elucidate the mechanism for houndary migration. Because orientation is the controlling factor SUIface-free energies may play a n important role. On the other hand, stress in the lattice may he responsible for this growth which then takes place to alleviate stress. This would not be inconsistent with the fact that orientation is important, as rsnisotropic crystds are elastically anisotropic and, therefore, one partioular direction might be expected to grow st the expense of another. Although the t N e mechanism may be very difficult t o determine with certainty, i t is at least true that two crystals having the same orientation do not show boundary migration. These studies may be impartant in metallurgy if they can be shown t o he related to recrystallization in metals. LITERATURECITED (1) Bryant, W. M. D.. J . Am. Chem. Soc.. 55,3201 (1933). (2) Gilpin, V.,et al.. Zbid.,70.208 (1948). (3) Kofler, L.. and KoEer. A , , "Mikromethoden zur Kenneeiohnung
orgenischer Stoffe und Stoffegernische," Innsbruck. Universititsverlag Wagner, 1948. (4) Lehmann, 0.. "Die Krystallanalyse." Leiwig. Engelmann. 1891. (5) MeCrone. W. C.. Srnedal. A,. and G i l ~ i n V.. . IND. EXG.C ~ E M . . ANAL.ED.,18, 578 (1946). (6) Mc4thews.F. W . , A N A LCHEM.. . 20, 1112 (1948). (7) Mitchell, John. Jr.,Ibid.. 21,448 (1949). (8) Tammann. G.. and Drever. K. L.. Z . anow. allom Chem.. 182.
289-313 (1929) R ~ o e w e oDecember 27. 1948. Work supported partly by fundamental re search spp~optiationby Armour Research Foundation and partly by grantin-%idfrom Resekroh Corwration. Based on work origindly submitted in partial fulfillment of requirements for Ph.D. in chemistry at Cornell University. 1942.
