826
ANALYTICAL CHEMISTRY
pictures are generally taken of ninhydrin-treated sheets: blue filter, no filter, red filter. The green filter may be used in place of the no-filter exposure if maximum contrast is desired. The following exposure times have been established: 2 seconds without filter, 60 seconds with the 49 filter, 25 seconds with the 61 or 29 filter. Films are developed and printed t o 2.75 X 4 inch size (2.5 times enlarged) by a commercial photographer, using an automatic printing machine. h file of these prints provides an objective, permanent, compact, and inexpensive record of all chromatographic runs. Figure 4 shows the photographic records of one-dimensional runs of twenty amino acids, ammonium chloride, and a protein h>drolyzate, developed with ninhydrin. Both rows of pictures show how the red filter aids in differentiation within the hydrolyzate chromatograms: The area a t R,60 is greatly “thinned” by the red filter, as are the methionine and valine spots on the same level, indicating their reddish color. The upper wries further shows that cysteine yields a primary reddish spot at R ~ 4 and 3 a smaller, gray-blue spot due to cystine. I n the lower wries it is evident that tryptophan is accompanied by two secondary spots, probably resulting from acid decomposition. The J ellow or brownish colors of proline and hydroxyproline are recorded by enhanced blackness in the blue-filter picture, and
near-disappearance in the red-filter picture. The relative intensities of the blue-filter and no-filter prints also suggest that the weak color shown by the hydrolyzate a t Rj45 is not due to hydroxyproline. ACKNOWLEDGRTENT
The authors are indebted to Richard J. Block for recommending the use of an aquarium tank with ascending-descending fl0W.
They are also indebted to the MacCallum Stores of Ardmore, Pa., for their friendly cooperation in the development of the photographic procedure. LITER4TURE CITED
(1) Consden, R., G o r d o n , A. H.. a n d M a r t i n , A. J. P., Bzochem. J . , 38, 224 (1944). (2) W i n e g a r d , H. M , , Toennies, G., a n d Block, R. J., Science, 108, 506 (1948).
RECEIVED December 1, 1950. Work supported in part by a research grant from t h e National Cancer Institute of t h e National Institutes of Health, United States Public Health Service, a n d by a n institutional grant of the American Cancer Society.
Analogous Nitro and Nitroso Compounds Separation, Identification, and Quantitative Estimation W. R . EDWARDS, JR., AND CILTON W. TATE Louisiana State Unicersity, Baton Rouge, La.
AKY reactions of nitrogenous organic substances, particularly oxidations, result in the formation of mixtures containing, among other things, analogous nitro and nitroso compounds. Difficulties are encountered 15 hen the separation of such coexisting products is attempted by the usual methods. Similarities in solubilities make the use of selective solvents unsatisfactory, and such circumstances as unsuitable vapor pressuies and insufficient stabilities may interfere M ith distillation procedures. These difficulties may reach prohibitive proportions when the desired products exist in small total quantity, in dilute solution, and in the presence of other substances. The present work describes a method for the separation of such compounds from solution and from each other by chromatography; for the partial identification of each by observation of the position and color (natural or developed) of its adsorption zone; and for the further identification and quantitative estimation of each by spectrophotometry. The method would serve equally well for analysis of a solution containing only one such compound The five pairs of C-nitro and C-nitroso compounds studied responded well t o both chromatographic and spectrophotometric treatments; it seems probable that many similar pairs would do the same. Study of one iV-nitroso compound indicated a possible extension of the method to mixtures of such related substances as nitramines and nitrosamines. The fact that extremely small quantities of solutes in very dilute solutions were separated and estimated with a t least approximate accuracy under mild conditions gave renewed evidence of the usefulness of such methods in work with compounds which are relatively unstable when heated or isolated, and particularly in the field of high explosives research. Trueblood and his coworkers ( 1 4 ) have employed similar techniques t o advantage in this specialized field. The present work was also approached a t one point by the papers of Gullstrom et al. (6),describing the separation and estimation of small amounts of p-benzoquinone monoxime (tautomei ically equiva-
lent to p-nitrosophenol) from mixtures containing also the dioxime and the by-products formed during nitrosation and oximation of phenol. Other uses of chromatography in conjunction with spectrophotometry have been described by various authors ( I , 2, 6, I d ) . APPARATUS
A KO.1 chromatographic column (9 X 130 mm.) was employed for most of the separations. It was filled with adsorbent to a height of 80 mm., using the technique described by Strain (IS).
Oxidations and other reactions of nitrogenous organic compounds frequently produce mixtures containing analogous nitro and nitroso compounds, whose separation and analysis by other methods are in some instances relatively difficult. The chromatographic characteristics of five C-nitro compounds, their C-nitroso analogs, and one N-nitroso compound were determined. All were recovered effectively from very dilute solutions by this method. Each nitroso compound was much more strongly adsorbed than its nitro analog, a circumstance favoring accurate separation. Standard spectrophotometric absorption curves were constructed, suitable for their identification and estimation. The combined chromatographic and spectrophotometric procedures offer a means of analyzing a solution containing minute quantities of a nitro compound and its nitroso analog, or either one. Specifically, the method has been applied to five such pairs; it appears probable that it could be extended to others. It should have special value in work with explosives.
V O L U M E 23, NO. 6, J U N E 1 9 5 1
827 first portion. It was then washed three times with 10% aqurou5 sodium carbonate and three times with water, dried over anh!-drous sodium sulfate, and distilled over sodium metal, the Ersction boiling a t 65' to 67O being retained for use. EXPERIMENTAL PROCEDURES
The chromatographic characteristics of the individual nitro and nitroso compounds (Table 111) were determined a' follows: -
Tahle 1. Name
200
210
220
2:O
240
250
260
270
W A V E L E N G T H IN
280
290
300
310
?,4-Dinitroresorcinol ?,&Dinitrosoresorcinol
PO
1-Sitro-%naphthol
Figure 1. Absorption of p-Yitrophenol and p-Nitrowphenol in 4bsolute Ethyl Alcohol
1-Nitroso-2-naphthol '2-Sitro-I-naphthol
In two specified inatimres. it S o . 2 column (20 X 220 m i n . , tv used. In the preliminary purifications of the nitro and nitro compounds, a No. 4 coluniii (48 x 300 mm.) was employed. Extinction coefficients were determined by use of a Rec.kninn Model DU spectrophotometer.
2-Sitroso-1-naphthol ?,l-Dinitropropane 2-Nitroso-2-nitropropane Diethylnitrosamine
;\ITERIALS
The solutrs obtaincid or prepared as indicated in Tahlcb I n ~ r e further purified chromatographically immediately before u about 0.4 gram of mat,erial in each run was treated with solven adsorbents (prewashed), and drvelopers ident,ical to those shon-n in Table 111. The appropriate zones were eluted Lvith absolute alcohol, and the solvent was removed by evaporation at r o o n ~ temperature. When Celite Lvaa used as a filter aid, the proportions were: 2 parts by weight of silicic acid to 1 part of Celite. The adPorbents were standardized (Table 11) hy determination of the R values, with respect t o them. of 0.6-1nl.portions of a 0.01 -11 solution of o-nitroaniline in btrnzenc~(method of LeRosen, 9). Such standardization permits conversioii of data obtained with one adsorbent system to other systems. A4dsorbents \?-err not prewashrd, except when this is mentioned specifically. In such instances it was accomplighed t>>employing, in succession, 1 volunle of acetone, 1 volume of ether, and 2 volumes of petroleum ether; "1 volume" is defined as the quantity whirl1 n-ould barely wet the entire column, so that ita top became dry just as the first liquid reached the hottom. The petroleuni r.thei. \vas purified as follows: Commercial petroleum ether R \~-:t* shaken with several successive portion? of fuming sulfuric acitl. standing overnight in contact Jvith the
Table 111. Compound p-Sitrorhenol
3leltiuq Point. C.
Eaatnian Iiodak Co.. He-earch Laboratory Synthetic. nitropation of phe-
p-Sitrosophenol
rfi
Reierencr
Source
Solutes p-Nitrophenol
1
>laterials
Solvents Benzene Ethyl alcohol iabaolute) Acetone Ether (anhydrous) Petroleum ether
nol
3rnthetic. two-sten nitration of renorcinol'l Eastrnan Kodak Co., Research Laboratory Synthetic. oxidation of i nitroso-2-naphthol with n ~ . tric acid Eastnian Kodak Co., Reqearch Laboratory P3-nthetic, oxidation of I-nitroso-1-naphthol with alkaline hydrogen peroxide Eastrnan Kodak Co., H e search Laboratory Synthetic, oxidation of 2-nitroso-2-nitropropane w t h chromic acidc Synthetic, nitroaation of ?nitropropane Synthetic. nitrosation of diethylamine l l w c k , reagent grade. t h i w phene-free t-,S. Induqtrial Cheiniral CII.. C.S.P. U e r c k . reagent grade. redistilled and dried Mallinckrodt Chemicai Works. reagent grade Pkellysolve Petrolerun Co.. Skellysolre B 9
Adsorbents Silicic acid Nerck. reagent grade .Jolins-1\Ianville Celite 2-Sitroresorcinol formed as intermediate. b Decomposed. C Much improved yield obtained by condiicting oxidation a t 15-15' f o r 24 hours, instead of using elevated ternperatlire described in reference. d Boiling point. e Purified; see text.
Table 11. Adsorbent Silicic acid Silicic acid-Celite Silicic acid-Celite (prewashed)
Standardization of idnorbents
Sollltlon 0 . 0 1 .+.o-nitroaniline I in benzene 0 . 0 1 .1f o-nitroaniline in benzene 0 . 0 1 .lf o-nitroaniline in benzene
Developer Benzene Benzene Benzene
R Vaiiii! 0 374 0 534 0 512
Chromatographic Characteristics of Nitro arid Nitroso Coinpounds
Solvent Benzene
Adsorbent Silicic acid a n d Celite
pNitrosopheno1
Benzene
Silicic acid a n d Celite
2,4-DinitroresorcinoI
Benzene
Silicic acid and Celite
2,4-Dinitrosoresorcinol
Benzenr
Silicic acid a n d Celite
1-Nitro-2-naphthol 1-Nitroso-2-naphthol 2-Nitro-1-naphthol 2-h7itroso-l-naphthoI 2,2-Dinitropropane
Benzene Benzene Benzene Benzene Petroleuni ether
Silicic acid Silicic acid Silicic acid and Celite Silicic acid a n d Celite Silicic acid
2-Nitroso-2-nitropropanr
Petroleum ether
Silicic acid
Developer R Value 0 378 Benzene (96%) Acetone ( 4 % j Benzene (96%) 0,222 Acetone (1%) 0.382 Benzene (96%) Acetone (4%) 0 .lOOb Benzene ( 9 6 % ) Acetone (4%) 0.894 Benzene 0.284 Benzene 0.960 Benzene 0.213 Benzene Benzene (50%) 0 850d Petroleum ether (50%) 0.416 Benzene (50%) Petroleum ether (50y0) Petroleum ether 0.060d noted, colors existing before streaking
Petroleum ether Silicic acid Diethylnitrosa mine Except where a Streaked with 6 .V S a 0 1 3 unless otherwise indicated. b Approximate value,, boundaries of zone relatively indefinite. Streaked with solution containing 0.005 M K M n 0 4 and 0.125 .If NaOH. d Approximate values, positions of zones determined spectrophotometrically. Streaked with diphenylamine in H1SOd. Boundaries indefinite and unreliable 1 Colorless when dry. color restored by streaking with benzene.
Color of Zone Before .4fter streaking streaking' Colorlew Yellow-
bensitli ltv [after otreaklngl Lf 0 00004
Colorlew
Tellou-
0 00008
Yellow
I-ellow
0 00008
Colorle-s
Green-blue:
0 0005
Yellow Red-brown Yellow Yellow Colorless
Yellow Red-yellow Yellow Yellou BlueC
0 00004 0 0002 0 00004
Bliiei
Bluet
0 0004
Colorleqs irere intensified by streaking.
0 00004
828
ANALYTICAL CHEMISTRY
Prelimina experiments were made to ascertain a combination of solvent, axorbent, and developer which would serve efficiently for the isolation of each compound. Where necessary, the rate of movement of an adsorbed zone was increased by dilution of the adsorbing silicic acid with Celite, or by dilution of the benzene usually used as the developer with acetone, or both. All initial solutions were 0.01 M except three containing solutes of relatively low solubility: p-nitrosophenol (0.0004 M ) , 2,4dinitrosoresorcinol (0.0025 M ) , and 2-nitrosc-2-nitropropane (0.0025 M ) .
i
0. I- NITRO- 2 - N A P H T C X C: '
3 13 A T 3 3 C m U
X
5 Y
~
-
4.5
2
k 8
40-
z
35
0 u
-
f 10Y x
$
I? Y
320
300 90
330
340
350
360
380
370
330 400
40
420
WAVE LENQTH IN my4
Figure 4. Absorption of I-Nitro-2-naphthol and I-Nitroso-2-naphthol in Absolute Ethyl Alcohol
I I CSO
I
1 150
I I70
I
I
I
I 110
I
310
WAVE L E N Q T H
Figure
I
I
290
I
I t a50
,,t P
j b NITROSO-2-NAPHTHOL f-
13.9 A T 2 6 O V
IN m,u
2. Absorption of 2,P-Dinitrosore~rcino~in Absolute Ethyl Alcohol
p 11 w
0
P L
WAVE LENGTH W m p
Figure 5.
Table IV. I
320
I
I
340
I
I
310 WAVE
Figure 3.
I LENOTH
I
380 IN
I
I
400
I
I
4LO
I
Absorption of I-Nitroso-2-naphthol in Absolute Ethyl Alcohol
Recoveries of IVitro and Nitroso Compounds from Solutions
mp
Absorption of 2,4-DinitroresorcinoI in Absolute Ethyl Alcohol
One milliliter of a solution of the compound undergoing examination was delivered to the top of the column. As ita upper edge disappeared into the adsorbent, addition of one volume of the developer was begun. The R value of the solute under the experimental conditions was determined. Streaking agents were employed t o produce or to intensify zone color. Similar runs were then made with increasingly dilute solutions to determine the sensitivity of the method for each compound-that is, the lowest concentration which would give a dependably visible zone. The zones of 2,Z-dinitropropane and diethylnitrosamine were colorlem, and no streaking agents were discovered which would indicate their boundaries with sufficient precision, though a solution of diphenylamine in sulfuric acid produced a recognizable but ill-defined blue shade with the former. The positions of these zones, therefore, were determined by systematic spectrophotometric examination of successive portions of the columns. Spectrophotometric data for the nitro and nitroso compounds were obtained from standard solutions in absolute ethyl alcohol, with readings at 5 my intervals. The elapsed time between final purification of each solute and its spectrophotometric measurement WRS made as short as possibk to minimiw errnre due t o
Compound o-Xitrouhenol &Nitrobheno1 pNitrosophenol pNitrosopheno1 2,4-DinitroresoroinoI 2.4-Dinitrosoresorcinoi 1-Nitro-2-naphthol
~2-Nltrono-l-naohthoi :$:,O~i'$~t~~~l
Conoentration, .M 0.01 0.01
0,00041 0.00041
0.01 0.0025 0.01 0.01 0.01 0.01 0,005
Quantity C h r o m a b Size of o/c graphed, bfl. Column Recovery' 97 No. 1 98 NO. 2 92 No. 1 98 NO. 2 NO. NO.
94
NO. NO. No.
94 91 95 93 88 79
KO.
NO. 2,2-Dini
