V O L U M E 25, NO. 6, J U N E 1 9 5 3 Table YI. .4nalysis of Lower iMolecular Weight Sulfonates NazSO4, % Weight Sample Designation Amperometrio Gravimetric (ASTM) 1A 0.31 0.30 2A 2.64 2.54 3A 1.20 1.21 0.70 0.72 4A 5A 1.86 1.84 1.16 1.12 6A 7A 1.72 1.44 0.35 0.37 8A 9A” 7.34 5.23 10.4 0.31 0.25 a Sample contained a n unusually large proportion of water-soluble sulfonates. On extraction of this material with 0.1 N NH4C104, most of the sulfonates entered the aqueous phase.
there should be a certain “area” of excess lead concentration wherein the desirable reaction was promoted while the undesirable one waq avoided. The fair agreement between the two methods for these sulfonates of low equivalent weight a t a certain concentration of titrant may be due to the fact that the gravimetric and amperometric methods are subject to the same general errors: the insolubility of lead sulfonates in the amperometric method and the insolubility of barium sulfonates in the ASTM gravimetric method. The observation that, where deviations occur, the am-
901
perometric method is usually higher indicate? that lead ip the greater malefactor. The use of concentrations of titrant different from those recommended would probably lead to gram deviations from the ASTM method when low equivalent weight sulfonates are analyzed, but not with the heaver sulfonates. This anomaly is due, in the authors’ opinion, to the chemistry of the different types of sulfonates and is not a general weaknws of the amperometric method. ACKNOWLEDG.MENT
The authors gratefully acknowledge the aid of R. C. Eiffert, who was concerned with the initial phases of this investigation, and W. H. Bruce, who assisted in the experimental work. LITERATURE CITED
(1)Am. Soc. Testing Materials, Conimittee D-2, Designation 85546T (1949). (2) Burdett, R. A., and Gordon, B. E., Ax.4~.CHEM.,19,843 (1947). (3) Heyrovskp, J., and Berezicky, S., Collection Czechoslou. Chem. Communs., 1,19 (1929). ( 4 ) Kolthoff, I., and Lingane, J. J., ”Polarography,” New York, In. terscience Publishers, 1941. (5) Kolthoff, I., and Pan, J. D., J . Am. Chsm. Soc.. 62, 3332 (1940). (6)Lingane, J. J., IND. ENG.CHEM.,ANAL.ED.,16,147 (1944). RECEIVED for review July 23, 1951.
dccepted l i a r c h 4, 1953.
Detection and Estimation of Impurities in Hexogen Chromatographic Methods EARL W. MALMBERG’, KENNETH N. TRUEBLOODS AND THOMAS D. WAUGH3 California Znstitute of Technology,Pasadena, Calif.
B a e a u ~the ptesenm of nitfamine impurities in the high explosive hexogen (hexahydro-1,3,5-trinitro-striazine) may affect its properties, a systematic procedure has been developed for their detection and estimation. The chromatographic properties and copreupitation characteristics of hexogen and a series of polynitramines of closely related structure have been investigated and a scheme for the concentration, isolation, detection, and approximate estimation of as little as a few parts per million of the different substances in hexogen has been de-
T
H E study of the mechanism of formation of the high explosive hexogen (hexahydro-1,3,5-trinitro-s-triazine) in the nitrolysis of hexamethylenetetramine has resulted in the discovery of a number of compounds of related structure which are possible intermediates and side products in the reaction ( 1 , ‘7, 15). The presence of some of these compounds even in small quantities in commercial production lots of hexogen may produce undesirable effects. The present report describes the development of a procedure for the detection and estimation of these and other possible contaminants in samples of hexogen. The nitramines which %-ere studied may be conveniently classified according to structure as follon-s. An identifying Roman numeral is given for each substance, together with the ab1 Present
address, Department of Chemistry, Ohio State University, Co-
lumbus, Ohio. 1 Present address, Department of Chemistry, University of California, Loa Angeles 24, Calif. I Present address, Arapahoe Chemicals, Inc., Boulder, Colo.
vised and tested. Accurate quantitative methods have been developed for the determination of hexogen and of octahydro-1,3,5,7-tetranitro-l,3,5,7-tetrazocine by chromatographic-spectrophotometrictechniques. Certain correlations between the structures of the different nitramines and their chromatographic and other properties are discussed. The general approach that has been used in this work shorld also be applicable to other mixtures of mmpounds which are closely related to each other in structure.
breviation by which the compound has sometimes been designated in both the classified and open literature. Compounds Containing a Six-Membered Ring I. Hexahydro-1,3,5-trinitro-s-triaeine(hexogen or R D X ) 11. l-Acetylhexahydro-3,5-dinitro-s-triazine(TAX) 111. Tetrahydro-3,5-dinitro-1,3,5,2H-oxadiazine (CyOx) Compounds Containing a n Eight-Membered Ring IV. Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine(HMX) V. l-Acetyloctahydro-3,5,7-trinitro-1,3,5,7-tetrazocine (SEX) One Bicyclic Compound VI. 3,7-Dinitro-1,3,5,7-tetrazabicyclo[3,3,1] nonane (DPT or DNPT) Open-Chain Compounds VII. 2,4,6-Trinitro-2,4,6-triazaheptane-1,7-diol diacetate (BSX) VIII. 2.4.6.8-Tetranitro-2.4.6.8-tetrazanonane-l.9-diol diace, , , ’ tate (AcAn) IX. 2(or 4)-acetyl-4(or 2), 6,8-trinitro-2,4,6,&tetrazanonane1,9-diol diacetate (H-16) X. 2,4,6-Trinitr0-2,4,6-triazaheptane-l,’?-diol dinitrate (ATX)
ANALYTICAL CHEMISTRY
902 Structural formulas of representative compounds are:
CHz
CEIi
02s-N
/
\
S-502
02K-N
/
AH2
\
Y-NOz (!XI$
tained on different columns and of standardizing the treatment of the adsorbent.
CH2-N-CHz
02s-N
/
/
\
\-
/
CH,
\
S--SO*
CH2-A-CHe
\-I
I n the developrncnt of the analytical methods described here, 'the chromatographic behavior of pure samples of the various nitramines was first studied, and specific procedures of separation and isolation were devised for each compound. If the analytical .procedure is to be suitable for the detection and isolation of trare amounts (0.1% or less) of the impurities, it is necessary t o concent r a t e the impurities in some manner first, as the limited solubility of hesogen prevents practical chromatography of more than about 50 mg. of this material a t one time on the usual column8. This preliminary concentration has been satisfactorily effected by fractional precipitation of most of the hexogen. Both the chromatographic and precipitation procedures have been test,ed extensively on known mixtures. The final analytical scheme provides both qualitative and approximately quantitative information concerning the presence of the various known possible contaminants, provided that they are present to the estent of a t least 10-4 to lo-%, and a t the same time permits the systematic detection and isolation of impuritiee of hitherto unknown structure. In addition to the scheme for the determination of minor impurities in hesogen( I), precise quantitative chromatographicspectrophotonietric methods for the estimation of I and IV have been devised; I T occurs to the extent of 8 to 10% in some commercial samples of I . These procedures provide a means of obtaining a positive check on the composition, and thus the purity, of samples of I. CHROMATOGRAPHIC PROCEDURES
Apparatus and Materials. The nitramines were supplied by W. E. Bachmann, A. T. Blomquist, and Marvin Carmack. The sample of X was prepared from VI1 in these laboratories by Philip Wilcox. The apparatus and general techniques used by the authors for chromatography on silicic acid have been described ( I O , 12). Chromatographic tubes of 19-mm. inside diameter, packed to a height of 150 mm., were used in all experiments except where otherwise noted. The adsorbent was a mixture of 2 parts by weight of hlerck reagent silicic acid q i t h 1 part of Celite 535. Uniform adsorptive properties and minimum spectrophotometric background were obtained by prewashing with acetone-ether, ether, and ligroine (saturated petroleum naphtha, 60" to 70') (14). Volumes of chromatographic solvents are given in the units of Vl50, where V,,, is the volume of solvent necessary to wet a 150-mm. column of adsorbent. The authors find this definition preferable to the previous (9) generalized definition of V as a quantity which is proportional to the length of the coluniii used. Because the ratio of the linear rates of movement of the zone and the solvent, R ( 8 ) , ip of considerable theoretical and practical importance, and the absolute distance a zone is developed by a given volume of solvent is independent of the length of the column, a unit of solvent volume which will represent a fixed absolute thtance of movement of the solvent front down the column, regardless of the length of the column, is convenient in chromatography. The present definition provides such a unit and thereby permits a simple means of comparing results ob-
All solvents except the absolute ethanol used for spectrophotometry were distilled in an all-glass still before use. Certain samples of the nitroparaffins contained an impuritj- (probably formaldehyde) which gave the same color reaction Kith the Schrv;er ( I S ) streak reagent as did some of the nitramines; percolation through activated silica gel (Davison Chemical Co.) and subsequent distillation was sufficient to remove this impurity.
Sample Solvents. The nitramines are appreciably soluble only in such solvents as the nitroparaffins, ethyl acetate, pyridine, and acetone, all of which are usually very strong developers or eluents. Fortunately, however, the nitramines are relatively strongly adsorbed on silicic acid and therefore these solvents can, by suitable admixture with benzene or ligroine (60' t o io"), be used to obtain compact zones of most of the compounds a t the top of the column. Mixtures of the nitroparaffins with benzene are the most suitable sample solvents found, but other mixtures are also of value, as will be seen upon inspection of some specific examples in following sections. -41 to 4 mixture of nitromethane and benzene is satisfactory for most of the compounds a t concentrations of about 1 mg. per ml. For the least strongly adsoibed compounds, X and 111,this mixture is too strong an eluent; fortunately, however, these compounds are more soluble than the others and can be dissolved in and chromatographed from less strong eluents, such as 1 to 4 dioxane-ligroine or 1t o 9 ethyl acetate-ligroine. Streak Reagents. The colorless zones of the nitramines on chromatographic columns may be detected by means of reagents applied in a streak along the extruded column. Chief reliance has been placed on two such tests, one designed for the detection of nitramino groups and the other for the detection of formaldehyde, ryhich is released by the decomposition of these compounds.
THE BENZENE-FRAXCHIMONT TESTis a modification of the Franchimont test for nitramines ( 4 ) ,which consists in treating the suspected nitramine with any one of several aromatic amines in the presence of zinc dust and acetic acid. The authors' extremely sensitive modification of this test, which was evolved empirically, is carried out by successively streaking the column with zinc dust (with a camel-hair brush), benzene, and finally the Griess reagent (2, 3, 5 , 6), a solution of 0.5$Z0of sulfanilic acid and 0.15% of 1-naphthylamine in a 30V0 solution of acetic acid in water. A positive test is indicated by the development of a deep red-pink color within the limits of the zone. -4s little as 0.01 to 0.02 mg. of most of the nitramines inveatigated can be detected with this test when the compound has been chromatographed so that it forms a zone in the upper part of a column 14 mm. in diameter. The columns should be postwashed with petroleum ether (saturated petroleum naphtha, 30' t o 60") before the benzene-Franchimont test is applied, as many of the common developing solvents interfere with the sensitivity of the test. The test is not specific for the nitramine group because the Griess reagent gives a pink color with nitrous acid; thus, for example, S-nitroso compounds also react positively. The niechanism of the benzene-Franchimont test with nitramines is obscure. Omission of any of the reagrnts decreases the qensitivity of the test considerably and may make it fail completely. The function of the benzene, which increases the sen-itivity by a factor of 3 or 4,is puzzling; it has been demonqtrateti that the thiophene which is present in ordinary commercial benzene is not responsible for the intensification which beniene produces. In the preparation of the Griess reagent, it is desirable to purify the 1-naphthJ-lamine chromatograp)ically. For this purpose, 1 gram of 1-naphthylamine is developed on a prewashed column with V ml. of 1 to 4 benzene-ligroine and the column is then postmashed with 1.5 V ml. of petroleum ether. The colorless zone of 1-naphthylamine occupies the upper third of the column,
V O L U M E 25, N O . 6, J U N E 1 9 5 3
903
naua11~~ lying between narrow zones of colored impurities: the amine may be conveniently eluted nith glacial acetic acid.
SL,IIRYVER TEST. Several of the nitramines derived from hexanicth\.Irnetetramine are readily decomposed by concentrated suliuric : i d to give formaldehyde, and the Schryver test ( 1 3 ) for ihis altlt.h\-de can therefore be applied. As modified for chroniarographic purpqses, the reaction is effected by streaking first with a 5 7 solution of phenylhydrazine in 90% sulfuric acid and then n-ith a SCC aqueous solution of potassium ferricyanide. Thc test is very insensitive in the presence of benzene; for best i,c-ults the column should be postu-ashed with petroleum ?:her. The Schryver test is about as sensitive as the heiizcwFranchimont test for 111, \-I, VII, and X and is only slightly leru seii.itire for VI11 and IS. On the other hand, I, 11, and 1,do not react with the Schryver reagent sufficiently rapidly for the Test to be practical. Developers. Exploratory investigation of the chroniatogtaphic properties of the pure nitramines with a wide variety of levelopere n-as necessary before appropriate condit,ions could he lourid for effecting the group separations which were a necessary preliminary to the final isolation and purification of the individual poesiiile impurities. The most generally satisfactory developers iound ai'e listed in Table I. Certain other solvent inistures which are suitable developers for specific separations are described belon-. I n general, a satisfactory developer is one which ,:levcJlopsa sample of a few milligrams a t a rate betxeen R = -a. 0.05 and R = ca. 0.4.,and which does not. cause escessive ~preatlingof the zone, double zone formation ( 9 ) , or any other .inonialous behavior. Some variation occurs in the adsorptive
Table I.
Satisfactory Derelopers for Hexogen and Related Sitramines on Silicic Acid-Celite"
C haractei istic Development b Volume, I'm nil.
Composition, Yo
A. 10
..
3
.. ..
1
B.
Compound
Com me n r
Development by Benzene I All other nitramines except 111 a n d S adsorbed above I11
X
I
Zone spread down from top of column rather t h a n developed
Development by E t h e r in Benzene S o t separable; concentrations of ether u p t o 50% develop them yerj' slowly 50 Yot separable; high concen33 trations of erher spread VI 30 I11 30 20 IX 6 8 Not completely separable
>;}
4 4
3
1
2 3 3
$;'}
C.
3 2
1:
12
2 2
8 7
2
D.
2
2 2 1 1
:.";X o t separable
25 2.5
2 2
2
Development by E t h y l Acetate i n Benzene 50 N o t completely separable
N o t separable
$';:}
Not separable
Development by Nitromethane i n Benzene N o t separable. concentra/:I tions u p t o 50% develop them very slonly 40 Xot separable 40 30 VI Xot completelj separable from VI1 12 IV N o t completely separable from V I 4 I 1 111
':;:,
' X i t h i n each group compounds are listed in order of decreasing adsorption .(fin:+..
1"""LJ.
6 Yolunie a n d composition of developer required t o move zone into middle third of column; composition is given as percentage by volume of first listed :more polar) component.
properties of different lots of silicic acid (14)even after prewashing, and the specific data presented here should be considered only as approximately valid for any particular lot. The relative adsorption affinities are, however, probably essentially independent of the lot used. Elution. The elution of each of the nitraminee from silicic acid-Celite was studied on a t least a roughly quantitative basis. A 1 to 1 mixture of acetone and ether is an effective eluent; as the iiitramines are nonvolatile. they can be recovered simply by evaporation of the bolvent. K i t h the particular lot of silicic acid (mixed with Celite) which nas used for almost all of the work repoited here, six of the compounds could be eluted essentially quantitatively. however, I, 11, IV, and 1- could be eluted only to an extent of between 90 and 95%. IV was selected for an exhaustive quantitative investigation; it was demonstrated that the percentage of 11- recoverable by elution under particular conditions Ma- ieproducible within 0.2 to 0.370, independent of variations in the size of the sample over the iange 1 to 10 mg. Coiicequently, it wai. possible to devise a precise analytical method for 11-,in spite of the impossibility of complete recovery, by applying an empirical but accurate correction factor; this method is described below. Attempts xere made to improve the recoverv by varying such factors as the volume of the eluent, 'tion of the eluent, and the time of standing of the adsorption romplex before elution, but theqe variations produced no more than minor changes in the percentage recovered. At a later time, hon ever, when another (and weaker) lot of adsorbent was available, it n a s found that the recovery of I was precisely quantitative. Consequently this new adsorbent was used in the development of the procedure for the quantitative estimation of I which is described below. There is evidence that IV could ako be quantitatively eluted with 1 to 1 acetone-ether from this weaker adsorbent, however, no careful study was made, as a pierise procedure for analysis for IV using the older adsorbent aaq available. Because 1-1 apparently undergoes either slow decomposition or slow irreversible adsorption on silicic acid-Celite, it is essential that this compound be eluted immediately after the completion of development. When this is done, the recovery is essentially quantitative; if, however, the adsorption complex is allowed to stand for even 10 minutes before elution, the percentage reroveiy falls below 90%, and after as long as 10 hours none can be recovered. -4lthough a similar effect was sought for each of the other compound; except X, in no other case did a detectable diminution in the recoveiable fraction occur when elution wadelayed even for as long as 16 hours. Severtheless, as a precaution, all zones were eluted immediately after development, with special attention to zones that might contain VI. CONCEYTRkTIOS O F IMPURITIES BY FRACTIONAL PRECIPITATION
Although chromatography is often an elegant method for the isolation of traces of one compound from large quantities of other substances, the method may by itself prove inadequate when all of the compounds are closely similar in solubility behavior and adsorption affinity. The need for a supplementary fractionation method is especially critical if the main constituent of the mixture is, like I, of very limited solubility; it is then difficult to chromatograph satisfactorily a sample sufficiently large to permit the isolation of a finite amount of the desired substance. Thus, in the present problem, some method of removing most of the I before chromatography is required. Extraction methods offer little promise in view of the similarities in the solubilities of all of the-e nitramines, and of the possibility that the impurities may be occluded in the crystals of I or even form mixed crystals with them. Although a procedure of concentration by partial crystallization of the I from solution might also be expected to suffer in part from the latter disadvantages, investigation proved that a practicnl method of this type could be developed. The fractional
ANALYTICAL CHEMISTRY
904 precipitation of the I, and consequent concentration of the impurities in the supernatant solution, were accomplished by diseolving the impure I in acetone and then precipitating the greater part of the I by the addition of petroleum ether. The material remaining in solution was readily recovered by evaporation of the solvent mixture. Detailed studies of the losses of each of the possible impurities during the procedure were, of course, necessary; the availability of the pure compounds made such studies possible by means of experiments with synthetic mixtures. Preliminary Considerations. .4 review of the chromatographic properties of the various nitramines showed that nitromethanebenzene was the best preliminary developer for separation of the possible impurities from I. Seven of the nine compounds are more strongly adsorbed than I with this developer; several milligrams of any of these substances can easily be separated from as much as 30 mg. of I on the usual column (19 mm. in diameter). On the other hand, the two compounds adsorbed below I (X and 111) can be separated well only if no more than 10 mg. of I is present in the final concentrate. Consequently, two concentration procedures and analytical schemes m-ere developed, one for the determination of the seven compounds adsorbed more strongly than I and one for the determination of those two adsorbed less Btrongly. Before the concentration procedures were devised, the approximate solubilities of the pure compounds in various acetone-petroleum ether mixtures were determined (Table 11). -4s preliminary tests had shown that the quantity of minor impurity was usually far less than 1%,the procedures were devised for and tested on mixtures containing 0.1% or less of impurity. The volumes and proportions of solvent were chosen so that there would be no loss of any impurity through its own insolubility, unless the amounts of it which occurred in the samples to be tested were abnormally large. If unusually large quantities of any impurity were found, a smaller sample of I could be taken originally with consequent simplification of the procedure.
Table 111. Recovery5 of Strongly Adsorbed Compounds from Binary Mixtures with 1000-Fold Excess of Hexogen dmount Recovered, Mg. 0.45 0.47 0.43 0.33 0.32 0.21 0.23
Initial .4mount, M g . 0 52 0 52 0 52 0 49 0 52 0 51 0 56
Compound
% Recovered 86 85 83 68
61
41 41
a From supernatant solution left after precipitation of most of I b y addition of 3.5 ml. of petroleum ether t o solution in 11 rnl. of acetone.
obtained when a mixture of 1.0 gram of I and 1.0 mg. of I11 was subjected to a double fractional precipitation, first from 15 ml. of acetone Kith 60 ml. of petroleum ether and then from 3 ml. of acetone with 24 nil. of petroleum ether. Procedure for Compounds Adsorbed More Strongly than Hexogen. -110-gram sample of I is dissolved in 150 ml. of warm acetone and partially precipitated by the rapid addition of 600 mi. of petroleum ether. After a short time the supernatant liquid is decanted and evaporated to dryness. The resulting residue is again fractionally precipitated by dissolving it in 15 t o 20 ml. of m-arm acetone and adding 4 volumes of petroleum ether. The residue from the evaporation of the supernatant from the second step is dissolved in 4 ml. of acetone and precipitated once more with 13 nil. of petroleum ether. The final supernatant solution is decanted and evaporated and the resulting crystalline residue is investigated by the systematic chromatographic procedure described below.
;
1 COLUMN C
Table 11. Compound
I
Solubilities of Nitramines in Acetone Petroleum Ether at 25" C. Solventu
A
1:3A-P 1 : 4 A-P 1:BA-P 1:8A-P
Solubility, Mg./Ml. Ca. 60 1.8 0.7 0.4 0.25
.~..-.
._____
1:8A-P >0.22 1:8A-P >o. 22 3:lOA-P Vb >O. 08 0 A acetone, P = petroleum ether (30°-60") ; composition given a s ratio of volumes. b Least soluble of compounds studied; consequently, solubility of all others in 3 : 10 A-P also exceeds 0.08 mg. per ml.
Vlll
X
Tests with Known Mixtures. A series of binary mixtures of 500 mg. of I with 0.5 mg. of each of the seven more strongly adsorbed impurities was used to test for losses inherent in the procedures of concentration (Table 111). The sample was dissolved in 11 ml. of acetone and most of the I was precipitated by addition of 35 ml. of petroleum ether; the supernatant solution was then decanted, and evaporated t o dryness. The impurity was isolated from the latter residue by a suitable chromatographic procedure and estimated spectrophotometrically. A second precipitation was necessary with the sample which contained IV, because a satisfactory chromatographic separation from the I present after the first precipitation could not be achieved. It was shown that, for all the compounds, mechanical losses and losses due to insolubility of the impurity itself were negligibly small. Experiments were done on the recovery of 111 from mixtures with I in order to test the reproducibility and the effect of small variations in procedcre. In general, the reproducibility wag surprisingly good; the results of duplicate experiments usually agreed within several per cent. A typical recovery of 60% was
.___._
COLUMN A
I11
-
_.--__
OF A L L
Z O N E A-2
1
F1LTRATE:THREE FRACTIONS c-l V l l l c-2 I X C-3 T
,
C -UMN B F I LTR AT E (l,lIl,X)
(REJECT)
FILTRATE: E-I (VI11,VII~
Figure 1. Schematic Diagram of Chromatographic Procedure for Isolation of Compounds Adsorbed More Strongly than Hexogen
----T.
Limits of zonsa Positions of cuts Teated for unknown compounds
V O L U M E 25, N O . 6, J U N E 1 9 5 3
905
Procedure for Compounds Adsorbed Less Strongly than Hexogen. The procedure for the less strongly adsorbed compounds is the same as that for the more strongly adsorbed conipounds, except that the third precipitation is made fiom 3 ml. of acetone a-ith 24 ml. of petroleum ether. Tests of Final Concentration Procedures. .Is the results in Table I11 indicate, the strongly adsorbed nitramines can be grouped into three classes according to the fraction recovered in the concentration procedure. Consequently, tests of the final form of the concentration procedure for these compounds 11ere made only with a representative compound from each of the three groups. Parallel tests were made with the less strongly adsoibed compounds. The results of the tests are presented in Tables I V and V. Comparison of the results in Table I V n i t h those in Table I11 shows that the three representative strongly adsorbed compounds chosen gave, respectively, the good, fair, and poor recorelies expected; the somewhat lower recoveries in Table I V are to be anticipated, as more precipitations mere made in the tests reported there. The results in Table V demonstrate that the recoverability of the less strongly adsorbed compounds is also approximately reproducible and substantially independent of the quantity present. The approximate reproducibility of rocovery in all these experiments indicates that rough correction factors can be estimated for calculating the initial amount of each inipurity from the quantity recovered. The implications of the losses which occurred are discussed below. CHROMATOGRAPHIC ISOLATION OF I M P U R I T I E S
Compounds Adsorbed More Strongly than Hexogen. The scheme by which the more strongly adsorbed compounds may be cliromatographically separated from a concentrate is shon-n in Figure 1. The experimental details of each chiomatogram are presented in Table VI. If the streak test is strongly positive on the chromatogram in which the final isolation of a given compound is accomplished, the streaked portion of the column is cut away, the zone eluted, and the nitramine estimated spectrophotometrically. If the streak iq weak or negative, the quantity present is too small for spectrophotometric estimation; consequently, the much more sensitive procedure of rechromatographing on a smaller column is used,
Table IV. Recovery of More Strongly Adsorbed Compounds from Binary Mixtures with a 4000-Fold Escess of Hexogen" Compound VI11 VI
I 1-
a
Initial Amount, Ng. 2 34 2 68 2 46
Amcunt Recovered, Rlg. 1.96 1.50 0.71
% Recovered 7s 5fi 29
10 grams of I ; three precipitations (see text).
Table V. Recovery of Less Strongly Adsorbed Compounds from Binary Alixtures with 2000-Fold t o 100,000-Fold Excess of Hesogena Compound
Initial .4mount, N g .
Amount Recovered. RIg.
5 Recoiered
I11
x T e n grams of I ; three precipitaticns (see text). Duvlicate exneriments Although it h a s b e e n r e orted by others t h a t X undergoes a transformaDion In acetone. as e v i d e n c e l b y a change in t h e melting point cf 9 when it is recrystallized f r o m this solvent, t h e authors have found t h a t t h e nieltinp point is never sharp, varying appreciably with t h e rate of heating, and t h a t X can be recrystallized from acetone without change in its chromatographic or spectrophotometric properties. b
and the amount of nitramine, if any, iq estimated by visual comparison of the streak color with that produced by known quantities of the same substance. This procedure of rechromatography on a small column should be applied to the residues from the evaporation of all nonspecific filtrates and of the eluates of sections of the column where no specific compound is expected to appear.
Table VI. Experimental Details for Chromatographic Isolation of 3Iore Strongly Adsorbed Compounds Dereloner
3 0 1 .i
2 0 1, 3
1.7
D
2.5 1.3 2.0
7 % S m in Be P 1 : 2 Et-Be P 15% EA in Re 14y0 Ac in Be P 6 % Ac in Be
1 3 1 Vo S APin Be 1 8 1 , .j P All samples placed on column from 1 . 4 S m - B e ; 0.3 Via0 used in chror~iatogram A a n d 0.15 V i ~ oin others. b See Fieure 1. C Compositions given a s ratios or percentages b y volume. Ac acetone' AA, acetic acid: Be, be,nzene; EB, e t h y l aoetate; Et, e t h e r ; P, betroleui< ether (30-60') ; Nm. nitromethane. d Fraction B-1 collected after 0.6 Viao of developer has entered column until 1.2 Viao of postwash has entered column. I n this a n d other columns all filtrates not collected for some specific purpose are investigated for preseiire of unanticipated nitrainino impurities. ' Column C is 180 mm. long. Fraction C-1 is collected from t h e time when 0.8 Viro of t h e ethyl acetate-benzene has entered the column until 0.7 Visa of acetone-benzene has enteied. C-2 is then collected until 0.2 VISO of postwash has entered column.
E
____
The reliability of the chromatographic procedure has been tested in several ways. A mixture of 26 nig. of pure I and 6 mg. of pure I V was put through the entire procedure; neither of these substances contaminated any of the regions in which zones of the other compounds were expected. The recovery of the compounds when they are present in quantities of about 1 mg. was tested experimentally by analyzing a mixture containing 30 mg. of I and 1 mg. of each of the strongly adsorbed compounds; the recoveries were: V, 82%; 11, 91%; IX, 96%; 1'111, 76%; VII, 86%; VI, 84%; and IV, 102%. These recoveries were considered satisfactory, as the aim of the scheme was to provide a qualitative and roughly quantitative analysis for traces of these impurities; undoubtedly the procedure could by suitable refinement be made more quantitative, if this end were desired. Because of the losses in the concentration and chromatographic procedures, the amount of a given substance which is isolated may be substantially less than the amount originally present. Approsimat,e factors for calculat,ing the amount of each impurity present in the original 10-gram sample from the quantity actually isolated can be estimated from the known losses in the isolation procedures, as these losses are approximately reproducible. The factors are: IX, 1.3; VII, 1.4; VIII, 1.6; V, 2.2; YI, 2.2: IJ3.5; and IV, 3 . 5 . Although these factors are not precise, they are entirely adequate for the desired purpose, the estimation of trace amounts. The over-all sensitivity of the analytical scheme ranges from 0.07 mg. for IT to about 0.03 mg. for I S in the original 10-gram sample of I. Surh very high sensitivity does have real significance as numerous test.* and checks on the procedures have demonstrated, although it i3 probably considerably higher than is necessary or even of practical interest. Compounds Adsorbed Less Strongly than Hexogen. The procedure for the chromatographic isolation of the less strongly adsorbed impurities is shonn in Figure 2. The espwimental tietails of each column are presented in Table J'II. The quantities of S and I11 iaolated are estimated either 11y spectrophotometry or by rechromatography, in the same mannc'r as with the strongly aclx~rbedconipoundP; in the rechromatog-
ANALYTICAL CHEMISTRY
906 raphy, care must be taken to choose the correct sample solvent. Checks were made on the analytical procedure both with pure I and with known mixtures and it was found to be entirely satisfactory. The correction factor for loss in the isolation procedure is approximately 1.8 .for each compound. The limit of sensitivity of the benzene-Franchimont streak reagent, about 0.01 nig. on a 14-mm. column, thus sets the limit of detection of either compound a t about 0.02 mg. in the original 10 grams of I.
-
Table \ l I . Experimental Details for Chromatographic Isolation of Less Strongly Adsorbed Compounds Sample Solvent Volume. Chromatogram" Vm ml. Campositionb FC 0 15 1 : 4 Np-Be
ZONE F-l
REJECTED
F I L T R A T E F-2(x)
A
L1
1
COLUMN J
-____
~
___..
J-2 (111)
----F I LTR ATE
Figure 2. Schematic Diagram of Chromatographic Procedure for Isolation of Compounds -4dsorbed Less Strongly than Hexogen
----S. T.
Ed
0 15
1 : 4 Nm-Be
,I
0 25
1 : 4 Py-P
1 : 4 Dx-P
1.5 1.5
2.2
1.5 1.5 1.5 1.3 1.3
Compcsitioii5 6% E.4 in Be
P
11% EA in
P
P
6 % Sin in Be
P
1 : l Et-Be
P
Compositions given as ratios or percentages by volume. Be. henzene: Dx dioxane: E.4, ethyl aretate; Et ether, P , petroleum ether (30-60'): S n ; . nitromethane: S p . 2-nitropropa1!1e; Py: pyridine Column F is 180 mm. long. S is washed through with sample solvent and first 0.3 1.150 of developer. Remainder cf filtrate is collected as usual a i d tested for unanticipated compounds. d Filtrate, containing I11 and some I , is collected after first 0.2 I - . ~ Iof de-elr,per has entered column. '1
OF A L L
I I
0 15
" See Figure 2.
i COLUMN H
F I L T R A T E H-3(l,lll)
c:
Developer Volume, VIXnil.
Limits of zones Positions of c u t s More strongly adsorbed compounds Tested for unknown compounds
Discussion. When as much as 0.1% of any of the impurities is present in the original 10-gram sample, several niilligranis of the material can be isolated in the chromatographic procedures and the identity can be confirmed in various ways-for elample, by melting point, spectral properties, or crystallographic characteristics. When the quantity isolated is only 0 1 nig. or less, the only evidence as to identity is provided by the chromatographic properties and color reactions; however, even then the identification may be made with some confidence, for the isolation procedure for each compound requires so varied a succession of chromatograms with different developers that there is very little chance that some unexpected substance will behave in precisely the same fashion as any known compound. QUANTITATIVE DETERMINATION OF HEXOGEN AVD OCTA-
HYDRO-1,3,5,7-TETRANITRO-1,3,5,7-TETRAZOCIXE
I n order to provide a check on the investigation of the purity of I by the methods described above, chromatographic-spectrophotometric procedures have been devised for the quantitative determination of I and IV in such samples. I t was not possible to elute these compounds quantitatively from all samples of silicic acid-Celite; the procedure presented here for 11' was devised and applied with a lot of adsorbent from which elution was not
quantitative. while that described for I was worked out with a different lot of silicic acid, one from which quantitative elution wa2spossible. The circumstances under which the work w3s done prevented investigating this interesting effect further. It is apparent that, as in all of the other chromatographic work reported here, the details of the procedures must be checked with the particular sample of silicic acid available. Procedure for I. -45-ml. aliquot' portion of a solutio11 of 50 nig. of dry I in 50 ml. of 2 to 3 pyridine-petroleum ether is diluted with 3 1111. of ligroine and transferred a t once to the prewashed chromatographic column with 8 ml. of benzene. The column is developed with 1.3 V I ~ml. O of a 470 solution of nitromethane in benzene and postwashed with 1.5 VIWml. of petroleum ether. The zone of IV (present to the extent of about 9% in certain Samples) usually extends from 12 to 22 mm. from the top of the column, the zone of I from 70 to 110 mm. The section of the column from 35 to 125 mm. is eluted with 150 ml. of 1 to 1 acetoneether; the contiguous port'ions of the rejected sections of the column are tested with the benzene-Franchimont reagent in order t~ detect any possible aberrations in the separation. The eluate is then evaporated to dryness under reduced pressure in an all-glass apparatus, 10 ml. of redistilled absolute ethanol is added and evaporated, and then the flask and ebullition tube are rinsed with a little acet'one. After the acetone has been evaporated in a gentle stream of pure air, t.he flask is evacuated with an aspirator and heated at 40" to 50" for 30 minutes in order to remove the last traces of volatile contaminants. Finally the crystalline rwidue is clissolved in 2.00 ml. of acetone and transferred to a 250-nil. volumetric flask with absolute ethanol; the comparison solvent for spectrophotometry is prepared in the same fashion. The corrected (11)optical density is measured at 235 mp; although there is no absorption maximum at this wave length, t'he slope of the curve is gentle and the position is satisfactory for the quantitative estimation. The value of C/D (11) a t this wave length ia 2.12 mg. per 100 nil. iilthough I shows an absorption maximum a t 213 nip, t,his wave length is not satisfactory for spectrophotometry after chromatography because of interference by impurities from the adsorbent. Discussion. The nitroparaffins, usually used in the *ample solvents for these compounds, cannot be used here because the resulting zone of I is spread too much. The pyridine-petroleum ether mixture gives a compact zone and has essentially no developing action. The percentage recovery of I from the adsorbent was tested with each phase of the chromatographic procedure. Uniformly reproducible recoveries of 99.6 to 100.0% were obtained: a small blank correction was necessary. The entire procedure was checked on known mixtures and was found to be precise and accurate to 0.2 to 0.3%. The same precision has been found in duplicate analyses of unknown samples. Procedure for IV in Presence of I. The procedure described here is for the determination of IV in a sample of about 25 nig. of I containing about 9% of IV-Le., for an absolute amount of IF' of about 2 mg. The procedure is, however, very flesible and may he adapted to samples of a wide range of sizes and coniposition?.
V O L U M E 2 5 , NO. 6, J U N E 1 9 5 3
907
I25-nig. sample of I is chroniatographed from 7.5 ml. of 1 to 4 nitroniethane-benzene; quantitative transfer to the column is completed with 8 ml. of benzene. The chromatogram is developed with 3.3 Vlsoml. of a 5qo solution of nitromethane in benzene and postwashed wit,h 1.5 Vlj, ml. of benzene and 1.5 V-160 ml. of petroleum ether. The zone of IV usually extends from 10 to 45 nini. and that of I from 85 mm. to 150 mm.; the upper 65 mm. of the column is elut’ed with 150 ml. of 1 to 1 acetone-ether. The Iow-er portion of the column is streaked in order to detect any aberration. The remainder of the procedure is identical with that for I , except t,hat the spectrophotometric estimation of the isolated 11-is made a t 228 to 229 nip; a t this wave length, C/D (11)for IV is 1.41 nig. per 100 ml. Discussion.
Because of incomplete elution it may be neces-
sary to apply an empirical correction factor to convert the quan-
tity of IV isolated to that. initially present. F o r the lot of adsorbent on which the procedure was developed, this factor was 1.115; a \vide variety of tests shoived it to be precise to about 0.2% and to Ire essentially independent of sample size over the range 1 to 10 mg. The precision and accuracy of the entire analytical procedure were tested with known mixtures and were found to l,e 0.2 to 0.3%; consequently, in a sample of I which contains iiljout 10% of IV the error will correspond to only about 0.0370 of the I. It has been found necessary to homogenize certain cmnmercial samples before reliable duplicate analyses can be idjtained on 25-nig. portions; this may be accomplished by dissolving 2 grams of the commercial product in 16 ml. of hot acetone, precipit,ating most of the dissolved material by the rapid addition of 75 ml. of petroleum ether, and finally evaporating the solvent in a gentle stream of pure air. After the residue has llren thoroughly mixed, it is dried in vacuo a t 61” and analyzed in the usual manner. GENERAL RE3IARKS
Correlation of Structure and Chromatographic Properties.
in the confirmation of other evidence as to the structure of certain of the compounds. For example, when this work was in progress, the linear structure of I S v a s considered proliable but had not been definitely established. The developmental characteristics of this compound, and to a lesser extent its behavior with the different streak reagents, afforded strong, confirmatory evidence of the correctness of the linear formulation. Losses during Precipitation Procedures. The losses of the different impurities in the fractional precipitation steps show interesting correlations with structure. Thus, among the more strongly adsorbed compounds, the three substances recovered in highest yield-VII, VIII, and IX-are linear and do not closely resemble I in structure, while of the other four compounds, all cyclic, the two recovered in lowest yield-IV and 11-are the most similar to I structurally. TI-ithout further data it is not possible to decide whether these facts a1.e merely a manifestation of a complex adsorption-occlusion process during precipitation, or whether some of the compounds may also form mixed crystals with I under these conditions. It has been reported that when crystals of I grow rapidly they have a strong tendency to occlude foreign material. Tests showed that coprecipitation did indeed occur in the experiments, and was apparently responsihle for the major losses. Evidence was obtained that adsorption on the surface of the freshly precipitated I might be responsiblc for a (comparatively small) portion of the loss: A solut’ionof 500 mg. of I and 1.OO mg. of 5’ in 10 nil. of acetone was treated with 35 ml. of petroleum ether and the resulting fine precipitate of I was rinsed with three 25-ml. portions of ethanol (in which I is essentially insoluble). The ethanol was found, after evaporation and chromatography, to have removed, presumably mostly from the surface of the precipitat,e, about 0.06 mg. of V, which corresponded only to some 15% of the total loss of V. It is apparent that the major portion of the loss is due to mechanical occlusion, mixed crystal formation, or both.
On
t lie hasis of t,heir general chromatographic behavior, hesogen and
the compounds related to it may be divided into several structural (.lasses. One of these classes, that of the linear nitraniino esters, iiicludes VII, VIII, I X , and X ; in comparison to the cyclic compouiitia t,his linear group is characterized by what may be termed a i~lativelyhigh sensitivity to ether-benzene developers-that is, small percentages of ether produce rapid development-and a relatively low sensitivity to the nitroparaffins. Within the group the adsorption affinity increases as the chain length increases, and also increases when an acetyl group is substituted for a nitro group on a nitrogen atom or an oxygen atom. The increase in adsorption affinity which accompanies the replacement of the nitraniino group by an .V-acetyl group is also evidenced in the cyclic compounds; thus, I1 is more strongly adsorbed than I, and V more than IT. In the cyclic nitramino compounds which contain no acetyl group the adsorption affinity increases with increase in the number of nitramino groups. The compounds in this group-I, 111, and IS’-are characterized by a sensitivity to developers that contain the nitroparaffins and by a relative insensitivity to those that contain ether. I1 and V. in which one of the nitramino groups has been replaced by an S-acetyl group, are extremely strongly adsorbed and show little or no sensitivity to particular types of developers. VI tends to show the strong adsorption affinity characteristic of the cyclic compounds. It is possible that a nitrogen atom a t a bridgehead, such as two of those in VI, may a t times be responsible for strong adsorption on silicic acid. Thus, hexamethylenetetramine requires aqueous developers for any movement a t all on this adsorbent. The above generalizations and classifications were of considerable help in devising the analytical schemes. I n particular, recognition of the varying sensitivities of the different classes to particular types of developers was of special value; examination of the schemes will indicate the way in which the information was used. A second useful application of the generalizations was
ACKKOWLEDGMENT
I t is a pleasure to acknowledge indebtedness to W A. Schroeder and Robert B. Corey, under whose supervision the work was carried out, to Linus Pauling, who was the official investigator, and to iirthur L. LeRosen, who helped in the development of the prewash and some of the streak tests. LITERATURE CITED
(1) Bachmann, W. E., and coworkers, J . I m . Chem. SOC.,71, 1842 (1949); 73,2769,2773,2775 11951). (2) Blom, J., Ber., 59, 121 (1926). (3) Feigl, F., “Spot Tests,” v 215, 2nd English ed., S e w York, Nordemann Publishing Co., 1939. (4) Franchimont, A . P. E., Rec. trau. chiin., 16, 227 (1897); Chem. Zent., 1897 11, 477. ( 5 ) Griess, P., Ber., 12, 427 (1879). (6) Hahn, F. L., and Jager, G., Ibid., 58,2335 (1925). (7) Jones, R. N., and Thorn, G. D., C a n . J . Research, B27, 828 (1949). (8) LeRosen, A . L., J . Am. Chem. Soc., 64, 1905 (1942). (9) Schroeder, W. A., Ann. A;. Y . Acad. Sci., 49,204 (1948). (10) Schroeder, W. A . , llalmberg, E. IT., Fong, L. L., Trueblood, K. N., Landerl, J. D., and Hoerger, E., I n d . Eng. Chem., 41, 2818 (1949). (11) Schroeder, W. A., R7ilcox,P. E., Trueblood. K. S . ,and Dekker. -4. O., ANAL.CHEX,23, 1740 (1951). (12) Schroeder,W. ii., Wilson, M.K., Green, C., Wilcox, P. E., Mills, R. S., and Trueblood, K. N.,I n d . Eag. Chem., 42, 539 (1950). (13) Schryver, S. B., Proc. R o y . Soe. London, B82, 226 (1910); Chem. Zent., 1910 I, 1366. (14) Trueblood, K. iK.,and Malmberg, E. W.,A N A L . CHEM., 21, 1055 (1949). (15) Wright, G. F., and coworkers, C a n . J . Research, B27, 218, 462, 469,489,503, 520 (1949). RECEIVED for review August 4. 1952. Accepted March 4, 1953. Contribution S o . 1718from the Gates a n d Crellin Laboratories of Chemistry. Based on work done for t h e Office of Scientific Research a n d Development under Contract OEAIsr-881 with the California Institute of Technology. Linus Pauling was t h e official investigator.
