2,4,7=Trinitrofluorenone as a Reagent for Microscopic Fusion Application to Polynuclear Aromatics and Some Substituted Benzenes DONALD E. LASKOWSKI, DONALD G. GRABAR', AND WALTER C. MCCRONE Department of Chemistry and Chemical Engineering, Armour Research Foundation, Illinois Institute of Technology, Chicago, I l l . This work was undertaken in order to develop a rapid method of identification of small quantities of polynuclear aromatics such as might be obtained as trace constituent? in a chromatographic separation. In this method, an addition compound is formed, using the Kofler microscopic mixed fusion technique. 2,4,7-Trinitrofluorenoneis used as a reagent for polynuclear aromatics. By the use of a simple microscope fitted with a Kofler hot stage, it is then possible to determine four significant melting points during one heating cycle. These are the unknown melting point, the addition compound melting point, and the melting points of the two eutectics formed on the mixed fusion preparation. This test has been applied to 30 polynuclear aromatics containing a variety of substituents and provides unequivocal identification of any one of these compounds. The method described in this paper can become a general method of identification of organic compounds, providing fusion reagents can be found that will react with a limited class of compounds.
A
idly established by measuring the significant temperatures enumerated above. To a large extent, the use of such specialized classification reaagents can be applied also to mixtures because the addition compound is usually quite insoluble and appears a t the zone of mixing even when the reacting component is present in very low concentrations. For example, this method has been applied to the study of composition of lubricating oil fractions obtained from chromatography. This method could be refined so that the morphological and optical crystallographic properties of the addition compound would be used to characterize the reacting unknown component in a mixture.
NOTABLE departure from the classical methods of identification of organic compounds has been the development of
microscopic fusion techniques, As carried on by the Koflers in Germany, identification of an unknown compound depends upon the measurement of the refractive index of its melt and the eutectic temperatures with two reference compounds (1). Fusion studies in this country ( 2 ) have proceeded along different lines. Most of the work has been directed toward detailed descriptions of the fusion behavior of individual compounds which serve for identification, alone or supplemented by crystallographic data. The method presented here represents the application of microscopic fusion techniques to the classical method of identification whereby a derivative is prepared, isolated, purified, and its melting point taken. I n this new extension of micro fusion techniques a derivative is prepared on a microscope slide, using a reagent which forms a molecular compound with a pure sample of the unknown. Then, without further isolation or purification, four physical properties of the compound are determined for the one preparation: the melting point of the unknown, the melting point of the derivative, the melting point of the eutectic between the unknown and the derivative, and the melting point of the eutectic between the derivative and the reagent. By choosing a number of reagents, each of which forms a molecular compound with a limited group or type of compound, a generalized identification scheme becomes possible. By this scheme, an unknown would be tested in a microscopic fusion preparation with each of the reagents until formation of a molecular compound is observed, This would immediately classify the unknown as a member of a definite chemical group. Identification of the particular member of that group could then be rap-
* Present address, Industrial Rayon Corp., Cleveland, Ohio.
Figure 1. Stages in the Preparation of a Mixed Fusion
The reagent used for the identification of polynuclear compounds is 2,4,7-trinitrofluorenone. Its use for this purpose has been reported by Orchin et al. (4), who have proposed that i t be denoted by the abbreviation TNF. I t forms molecular addition compounds with many polynucl~arcompounds, as well as with a few others which possess phenyl groups joined through a conjugated linkage and lying essentially in the same plane-e.g., stilbene and axobenzene. The technique used in making the fusion preparation is illustrated in Figure 1. -4small amount (about 1 to 5 mg.) of 2,4,7trinitrofluorenone is melted between a half slide and cover glass and allowed to so1idif.y (Figure 1, A and R). About the same quantity of the unknown is then placed a t the opposite edge of the cover glass, melted, and allowed to run under the cover glass into contact with the reagent (Figure 1, C and D). Finally the preparation is reheated, leaving a small amount of the unknown unmelted, and then allowed to resolidify. If the preparation is observed during this final crystallization the molecular compound and the two eutectic regions can be easily seen as they crystallize
V O L U M E 25, NO. 9, S E P T E M B E R 1 9 5 3
1401
successively (Figure 1, E), The preparation of the mixed fusion requires about 1 minute. The mixed fusion prepared in the above fashion contains areas of composition corresponding to each of the significant points a i the two-component phase diagram, shown for the general case in Figure 2. From one side of the zone of mixing to the other there is pure 2,4,7-trinitrofluorenone with melting point TI,eutectic between the reagent and the molecular compound with melting point T,,molecular cornpound with melting paint T,,eutectic between the molecular cornpound and the unknown with melting point T,,and finally pure unknown with melting point T,. Usually a single heating cycle on the microscope hot stage is sufficient to determine each of these temperatures in sequence.
per minute a t the melting point.
An excellent hot &@e designed the Koflers is available in this country i r o n A. E. Thomas Philadelphia, or W. J. Hacker, New York, N. Y. All of the compounds used in this study were purified either by recr stitllization or by mblimation and are believed to be a t lea& 0 pure. An attempt was made to determine the effect of small quantities of impurity on the melting point data in Table I. b
c".,
994
_. TNF
4
UNKNO"
P E R CENT U N K N O W N
Figure 2.
Simple Two-Component Phase Diagram Showing Compound Formation
The typical behavior upon heating of such a preparation is illustrated in Figure 3 which shows a series of photomicrographs, taken with ordinary light, of a 2,4,7-trinitrofluorenoneatilbene fusion. The molecular compound is the dark hand extending through the center of the preparation, bounded on either side by areas of eutectic composition. The reagent is a t the bottom of the picture, stilbene at the top. Upon heating, no change occurs until the eutectic between the molecular compound and the stilbene melts a t 116.4' C. leavine a thin line a i melt seoaratine the iwo as shown in Figure 3, B. At 124' C. the stilbene melts and the entire upper half of the preparation becomes light as in Figure 3, C. At139.2 ' C. the eutectic between the molecular compound and the reagein t melts, leaving the molecular compound as a strip of bright red-form a peritectic with the addition compound. The scheme works as well, of course, in either case; if the possildity of a peritectic between the unknomm and the addition compound is being investigated, 2,4,7-trinitrofluorenone should replace the word unknown and vice yersa in the following procedure. Approximately 1 part of 2,4,7-t.rinitrofluorenone and 2 parts of unknown (approximately 100 mg. in all) are t,horoughly mixed on a microscope slide and fused together. After eolidification a portion of the sample is melted quickly on the hot stage. The composition of the mixture should be corrected, if necessary, to make certain the addition compound d l be in escess when tl.z eutectic melts. The mixture is then purified using the absorption technique of the Koflers ( 1 ) . This consists of placing a small piece of filter
V O L U M E 25, N O . 9, S E P T E M B E R 1 9 5 3 paper on a microscope slide, p!aoing the powdered material on the paper, and then covering m t h a second half slide. The slide is then placed on the hot stage maintained a t a temperature above the melting point of the normal eutectic, and the top half slide is ressed down until the molten eutectic that forms is absorbex by the paper. This is repeated several times with fresh paper as the temperature rises alniost to the melting point of the addition cornpound. The umnelted residue is pure addition puu"". corn-----'
A portion of the purif ied addition COInpound is then mix:d ne with an rtppraximately e(p e l amount of 2,4,7-t~initroRuoren~~
1403 and the temperature of first melting is obsorvod. If this temperature coincides with the micro meking point of the purified addition compound, then the compound actually undergoes a peiiteotio renetion. If not, of course, a lower melting euteotic between the two components should be observed. Confirmation of the periteotic reaction is obtained by not,ing that the crystals of reagent grow during dissociation of the addition compound. The temperature listed in Table I in the space reserved for melting point of the addition compound is the peritectie resetion
ANALYTICAL CHEMISTRY
1404 temperature for those systems showing a peritectic. Each such case is indicated by a (P) after the temperature listing. Several cases of polymorphism are shown in Table I. The 6methylquinoline-2,4,7-trinitrofluorenone addition compound undergoes a solid-solid polymorphic transformation when heated to about 50" C. The o-hydroxydiphenyl-2,4,7-trinitrofluorenone addition compound, when allowed to form a t a temperature a few degrees below its melting point, forms two polymorphs, each of which undergoes a peritectic reaction. Their dissociation temperatures are measurable. The same is true of the 1,Zbenzanthracene addition compound, except that each polymorph melts normally. The addition compound of 2-naphthol undergoes a solution phase transformation at about 182' C.
8
4
:[
Because of the high melting point of naphthacene, the procedure for formation of a mixed fusion preparation as previously described was modified somewhat for this compound. Naphthacene was placed on a microscope slide under a cover glass, and a small amount of reagent was melted and allowed to flow under the glass until it came into contact with the solid naphthacene. After allowing to stand about 1 minute a t approximately 180" C., the slide was cooled and the eutectic and peritectic temperature were measured. All melting points reported in Table I are an average of a t least three separate determinations, and all values are reproducible to within f0.5' C. when carried out by the same operator with the same procedure and the same equipment. For this reason, the melting points are reported t o the nearest 0.1' C. However, deviations from these values should be anticipated for reasons discussed below. The values enclosed in parenthcses are the melting points of addition compounds as taken from the literature. The agreement is good with the exception of 20-methylcholanthrene and carbazole. The addition compound with 20-methylcholanthrene decomposed on heating; howevcr, the value reported is reproducible. No explanation can he offered for the difference in the reported melting point of the carhnxole-addition compound, In general, however, differences in melting point can be ascribed to the fact that the method of determining melting points varies considerably from labolatory to laboratory. Examination of Figure 4 shows the striking differences in a p
pearance of most of the addition compounds. These photoniicrographs were taken with ordinary light at room temperature. DISCUSSION
The melting points reported are accurate only under the conditions specified. Anyone using the melting point data in Table I should first determine the conditions which, with his equipment, will give closely similar values on two or more of the systems listed. Because of the larger errors introduced by impurities, different heating rates, and different e uipment, other analysts will probably obtain slightly different v3ues. However, discrepancies greater than f1%will seldom, if ever occur. Considering these rather conservative limits, it appears from Table I that three pairs will not be sufficiently differentiated: 1-bromonaphthalene and 1-iodonaphthalene, azobenzene and diphenyl, and p-aminoazobenzene and stilbene. In many cases, the use of simple supplementary tests such as color and refractive index should serve to differentiate between the components. In any case of ambiguity, it is advisable to carry out a second mixed fusion between the unknown and one or both of the possibilities. If the two compounds are different, a discontinuity in crystal shape, birefringence, color, and rate of growth, will become evident in the zone of mixing. If identical, of course, there will be no zone of mixing. The existence of several pairs of compounds whose melting point relationships are very close raises the question as to whether the four temperatures involved are actually independent. Figure 5 shows the melting point of the addition compound t o be completely random with respect to the melting point of the corresponding original compound. The same figure also shows that although the eutectic temperatures involved do not possess the same degree of randomness, it is sufficient to distinguish all but a few of the compounds involved. Of the 30 compounds reported, four are not, strictly speaking, polynuclear aromatics; these are azoxybenzene, azobenzene, paminoazobenzene, and stilbene. All of these compounds have a double bond in a position to conjugate with the benzene ring, and this perhaps accounts for the formation of addition compounds. Work now in progress further indicates that in certain cases substituents on a benzene ring, such as hydroxy, alkylamino, arylamino, and halo groups, permit the formation of addition compounds from th& melt. Furthermore, several of the comDounds reported in Table I are actually polynuclear heterocyclics containing nitrogen in the nucleus. From the results obtained so far, it is safe to say that the method of i lentification outlined in this paper is applicable to both condenseJ carbon and condensed heterocyclic systems. However, the effects of substituents on the heterocyclic rings would be different from the effects of the same substituent on a homocyclic condensed system. For instance, addition compounds are obtained from both quinoline and naphthalene. An addition compound can also be formed from I-nitronaphthalene, but not from 8-nitroquinoline, or l&dinitronaphthalene. This method can undoubtedly be extended to other groups of compounds by the use of different reagents. For instance,. picric acid would form addition compounds with amines. It is also likely that some type of Diels-Alder reagent woul I work with systems possessing conjugated double bonds. In all likelihood, additional reagents could be chosen from those already reported in the literature so that this procedure could be extended to include a large number of organic compounds. ACKNOWLEDGMENT
The authors gratefully acknowledge the assistance of Dorothy Baranski in preparing the photomicrographs used in this paper. LITERATURE CITED
(1) Kofler. L.. and Kofler, A,, "Mikromethoden zur Kennseichnung organischer Stoffe and Stoffegemische," Innsbruck, Univ. Wagner, 1948. ( 2 ) McCrone, W. C., Mikrochemie uer. Mikrochim. Acta, 38, 476. (1951). (3) Orchin, Milton, Reggel, Leslie, and Woolfolk, E. O., J . Am. Chem. SOC..69,1225 (1947). (4) Orchin. Milton, and Woolfolk, E. O.,Zbid., 68,1727 (1946). RECEIVED April 9, 1952. Accepted June 18, 1963. Presented before the All-Day Chemical Conference sponsored by the Chicago Section of the AM~RICA CHEMICAL N SOCIETY November 23, 1951, Chicago, 111.
