of acids, and of a n acid with a neutral internal standard, coupled with the failure of the formic acid-water injection to displace acids, all indicate that the responses observed are in fact due to the entire sample and therefore quantitative. LITERATURE CITED
(1) Ackman, R. G., Retson, M. E.,
Gallay, L. R., Vandenheuvel, F. A., Can. J. Chem. 39,1956 (1961). (2) Dahmen, E. A. M., Chim. Anal. ( P a r i s )40.430 (1958). (3) Emery, E. k - K o e r n e r , W. E., ANAL.CHEM.33,146 (1961). (4) Erwin, E. S., Marco, G. J., Emery, E. M., J . Dairy Sci. 44,1768 (1961). (5) . , Ettre. L. S.. in “Gas Chromatoeraphy,” 3rd International Symposium, p. 307, N. Brenner, J. E. Callen, M. D. Weiss, eds., Academic Press, Kew York, 1962. (6) Fukui, K., Nagatomi, H., Murata, I
,
S., Bunseki Kagaku 11, 432 (1962); C.A. 57, 978b (1962). (7) Gehrke, C. E., Lamkin, W. M., J. Agr. Food Chem. 9, 85 (1961). (8) Hankinson, C. L., Harper, W. J., Mikolajcik, E., J . Dairy Sci. 41, 1502 119581. (91 Hunter, I. R., J. Chromatog. 7, 288 (1962). (10) Hunter, I. R., Ng, H., Pence, J. W., Ibid., 32, 1757 (1960). (11) Hunter, I. R., Ng, H., Pence, J. W., J . Food Sci. 26, 578 (1961). (12) Hunter, I. R., Ortegren, V. H., Pence, J. W., ANAL. CHEM.32, 682 (1960). (13) James, A. T., Martin, A. J. P., Biochem. J . 50, 679 (1952). (14) Jowett, P., Horrocks, B. J., Nature 192.966 (1961). (15) Karmen, A.; McCaffrey, I., Bowman, R. L., Ibid., 193, 575 (1962). (16) Knight, H. S., ANAL. CHEM.30, 2030 (1958). (17) Kumeno, F.. J . Aar. Chem. SOC. ’ J a v a n 36. 181 (1962). ” (18) ‘Lovelick, J: E., ‘ANAL. CHEY. 33, 162 (1961). (19) McInnes, A. G., in “Vapour Phase
Chromatography,” p. 304, D. H. Desty, C. L. A. Harbourne, eds., Butterworths, London, 1957. (20) Metcalfe, L. D., Nature 188, 142 119601. (21) Prevot, A,, Cabeza, F., Rev, Franc. Corps Gras. 8,632 (1961). (22) Ralls, J. W., ANAL.CHEM.32, 332 (1960). (23) Raupp, G., Angew. Chem. 71, 284 (1959). (24) Saroff, H. A., Karmen, A., Healy, J. W., J. Chromatog. 9,122 (1962). (25) Smith, B., Acta Chem. Scand. 13, 480 (1959). (26) Smith. E. D.. Gosnell. A. B.. ANAL. ‘ CHEM. 34,438 (1962). ’ (28) Ibid., (27) Swoboda, p. 646.P. A. T., Chem. Ind. (London) 1960, 1262. (29) Wilkens Instrument and Research, Inc., rlerograph Research Notes, Summer issue, 1961. (30) Ibid., May 1962.
RECEIVEDfor review August 31, 1962. -4ccepted February 18, 1963.
Paper Chromatography of Substituted Trinitrobenzenes DAVID M. COLMAN lawrence Radiation Laboratory, University of California, livermore, Calif.
b A rapid classification of the dominant chromatographic functional group in 18 substituted trinitrobenzene compounds can b e made from R p values by using two paper partition chromatographic systems. Symmetrical trinitrobenzene is used as a reference for the functional group classification. Supplementing the RF data with the results of various heat, light, and spray tests permits a complete identification of any one of the 18 compounds.
I
AN EARLIER communication (1) the chromatographic spectra of 14 substituted trinitrobenzenes in 10 different solvent systems were reported. I n this report a chromatographic method using two solvent systems, derived from the 10 systems studied, is described which, when coupled with simple heat, light, and spray tests, permits the identification of any one of 18 substituted trinitrobenzene compounds. The use of lJ3,5-trinitrobenzene as a reference allows a degree of classification as to the dominant chromatographic functional group in the compound from the RF value. Generally, special tests are required for the final identification of the compound. This procedure has been of value in the rapid identification of explosive
components. It has also served as a method for determining the purity of samples of any of the 18 compounds.
17174th St., Berkeley, Calif.). Spraying apparatus. Streaking and self-filling pipets. Long wave a n d short wave ultraviolet sources. Whatman S u m b e r 1 chromatographic paper, 18- x 21-inch sheets. Reagents. Polar immobile solvent, 25y0 formamide (Eastman Kodak White Label) in acetone (v./v.). This solution should be made just before
EXPERIMENTAL
Apparatus. Chromatographic tanks are of a n all glass construction. Impregnating troughs are all glass construction (Kensington Scientific Co.
N
652
ANALYTICAL CHEMISTRY
U
0
O,’t-
U
0
0.0
‘ 1 A U 1 1 1 I PH3 PH2 PH
P
OH
(OH)3
(OH02
PH
PH
PH
NH2
OCH3
CH3 COOH
NH2
OH
OH
I
PH2 PH2 PH
I
1
PH2 PH
1
1
’
1
P
P+
PH
P
(OCH312 OW3
(NHJ2
1 P$ CI
C13
(CH3)2 CH3
I PH2 ”CH3
(CH&
“‘2
”2
Compound
Figure 1. RF values of substituted trinitrobenzenes with formarnide- [cyclohexane/ benzene (1 / 1 ) ] system
Color Results from Various Detection Methods
Table 1.
Detection method (DM-)G Compound
2
1
Cb
Y Y Y Y
1C 1Y Y C
Y Y Y Y
C C C
C
cC Pink 0
C
3
4
-
++ +++ +Y ++ ++ ++-
+++ ++ ++ Y ++ +++ ++
+
+
5
Gr P YO
YYYO
-
Pink
-
Y
6
7
8
Q Q Q Q Q Q Q 1Q Q Q Q Q Q Q Q Q Q
RO 0 Bright Y YO YYO YBr RBr YBr -Y YO 0 Y 0 Y Br Tan
0 RO YO YO
9
0
-
YO RO YO -Y Y 1Y 0 Brick R R R -
Br
0
Brick R YO
-
0
10
Q Q Q Q Q
i
P
-
Q Q Q Q Q Q
Brick R
Q
0
8
DhI- 1. Self color. 2. Drying on hot plate. 3. Long wave ultraviolet light. 4. Short wave ultraviolet light. 5 . Fiveminute exposure to short wave ultraviolet light and examination of visible light. 6." .V,iV'-diphenylbenzidine plus long wave ultraviolet light. 7 . c 1 X-NaOH and dry on hot plate. 8 . c 1 M-NaOH, dried on hot plate, then sprayed with Griess reagent. 9.c Di-n-butylamine-dimethylformamide. 1 0 . c Rhodamine B plus short wave ultraviolet light. b Color Code. C, colorless. Y, yellow. Gr, gray. P, purple. Q, quenching. 0, orange. Br, brown. R, red. (-), no change from original color. (,--), dark sppt. Reagents are applied by spraying.
using as it has poor keeping qualities. Polar solvent system, cyclohexanebenzene 1 to 1 (v./v.). Reversed phase immobile solvent, 10%)heavy mineral oil (Squibbs) in n-hexane Reversed phase solvent system, ion exchange water. Spray Reagents. Dhl-6, acetone saturated with Il',?T'-diphenylbenzidine. DM-7, 1M aqueous sodium hydroxide. DM-8, Crriess reagent (2), prepared by mixing equal volumes of a 1% solution of sulfanilic acid in 30% acetic acid with a O.l~o solution of 1-naphthylamine in 3Oyo acetic acid. Mix just before using. DM-9, di-nbutylamine-dimethylformarnide 80 to 20
(v./v.). DM-10, 0.10% solution of Rhodamine B in 4y0 aqueous hydrochloric acid (3). Procedure. T h e chromatographic tanks are prepared as follows: T h e cyclohexane-benzene t a n k is lined half way around from t o p t o bottom with heavy filter paper (Whatman 17 or 31ET). Two hundred milliliters of t h e solvent is placed i n t h e bottom. T h e water t a n k is not lined and contains 200 ml. of water at t h e bottom. The chromatographic paper is cut into 37- X 10.4-cm. sheets with the long dimension paralleling the machine direction of the original sheet. Lightly
3 OH
OH)^
(0145
CH3 N H ~
OH
NH2
COOH
OC% (OCH312 ( N H ~ ) ~ CCH~ CI
OH
KH3h
C13
CH3
(CH3)J
NfH3 NO2
"2
Compound
Figure 2. system
RP value:; of substituted trinitrobenzenes in the mineral oil-water
pencilled lines are drawn at 4 cni., 9 cm., and 34 cm. from one end, for the fold line, starting line, and front line, respectively. The sheet is divided into 1.9-cm. intervals, across the width of the paper, on the 9-em. line, thus allowing 5 compounds to be spotted and chromatographed simultaneously. The RF data shown in Figures 1 and 2 was obtained from this type of paper. If one wishes to apply more than one detection test to the same sample, it is better t o streak the sample across the paper a t the starting line. After chromatographing and drying, the sheet of paper may be divided into as many strips as needed for the various detection methods. Impregnating solution is put into the trough and the paper is drawn through with a slow steady motion. The paper is hung to dry a t ambient temperature. The polar phase paper is allowed to airdry for 15 minutes; the reversed phase paper is dried for 5 minutes. After drying, the paper is either spotted or streaked with a 1% acetone solution of the sample, (in one or two cases a saturated acetone solution is used because of solubility conditions). If the paper is being spotted, a 1.0-X or 2.0-h sample (depending upon solubility of the sample) is placed on one of the marked spots. After the acetone has evaporated from the placed sample, the paper is put into the proper tank and allowed to equilibrate for 30 minutes. Ten milliliters of solvent is then put into the trough and allowed to descend to the 34-cm. mark. The average time for the cyclohexane-benzene is 86 minutes; the time for the water is 130 minutes. When the front has reached the 34-cm. mark, the paper is removed and hung in a hood to allow the solvent VOL. 35,
NO. 6. MAY 1963
653
to evaporate. At this time the RF values can be determined and the necessary detection methods can be applied. The method of chromatographing is that of the descending one-dimensional type. The detection methods and results are shown in Table I. DISCUSSION
To avoid writing long formulas the folIowing notation has been adopted:
NOz is given the symbol P, thus, 1,3,5trinitrobenzene is written as PH3, 2,4,6-trinitrophenol is PH2 (OH), 2,4,6trinitro-3-methoxy aniline is PH(OCH3)( N H J , etc. In the formamide, cyclohexane-benzene system (Figure 1) it is immediately
seen that the compounds containing acidic groups have zero RF and those with the -NH2 group have a mobility less than that of PH3. The one compound with a nitramine group, PH2N(CH3,N02), also has a RF just below PHa. The remaining compounds have RF values above PHa. For the simply substituted compounds in this group, the RF order is CH3 > C1 > OCH2. When doubly or triply substituted, however, they all approach the same RF. Further differentiation may be made among the heavily substituted compounds by eluting and re-chromatographing in one or more of the systems previously described (1). Figure 2 shows the RF values in the reversed phase system, mineral oil, water. In this system there is a reversal in the RF values, all the phenolic group compounds having an RF greater than that of PHa. In the formamide system it is difficult to separate PHzCHs from PH(CH& and P(CHJ3, whereas in this system PHzCH3 can be separated from the other two compounds. Too, the
simply substituted compounds PHzCl and PHzOCHa are easily separated from their doubly- or triply-substituted counterparts. The figure also shows that in the aqueous system all compounds with an RF value less than that of PHs will not be detected readily if PH2NHs or PH2N(CH3,N02)are present, because of the long tailing of these latter compounds. Again, extraction or elution of these compounds and re-chromatographing in another system or systems will allow further differentiation. LITERATURE CITED
(1) Colman, D. M., J . Chromalog. 8, 399
(1962).
(2) Feigl, F., “Spot Tests in Organic Analysis,” p. 164, 6th ed., Elsevier Pub-
lishing Company, New York, 1960. (3) Feigl, F., Gentil, V., ANAL. CHEM. 27, 432 (1955).
RECEIVED for review September 6, 1962. Accepted February 8, 1963. This work was performed under the auspices of the U. S. Atomic Energy Commission.
Gas Solid Elution Chromatography with Graphitized Carbon Black C. G. POPE Department o f Chemisfry, University o f Otago, Dunedin, New Zealand
b The use of graphitized carbon black adsorbents allows the preparation of gas solid chromatography (GSC) columns of low HETP and high resolving power. Because of the high specific surface area, and surface homogeneity of these materials, the linearity of the adsorption isotherms is maintained up to relatively high surface concentrations, so that symmetrical elution peaks can be obtained on the chromatograms of even quite strongly adsorbed substances. The performance of these columns compares favorably with that of a n-hexadecanecoated capillary for the separation of hexane isomers, and allows the resolufrom which tion of a sample of CTF~~H, only one peak was obtained with the latter column, into four components. Their performance also appears to be superior to that of the GSC columns employing a “tailing reducer.” The reproducible nature of the graphitized carbon surface should allow relative retention times in GSC to be used as a method of qualitative analysis in the same way that they are used in GLPC.
654
ANALYTICAL CHEMISTRY
G
elution chromatography has been used very little as a n analytical method, except for the separation of volatile gas mixtures, where no suitable liquid stationary phase is generally available. The main reason for this appears to be the occurrence of very asymmetrical elution peaks with the majority of systems which have been studied. The reason for this asymmetry is the curvature of the adsorption isotherm. If only fairly small degrees of surface coverage are considered, this curvature may usually be associated with the occurrence of heterogeneous adsorption sites on the solid. Two general ways may be used to overcome this difficulty. If adsorption leads to only extremely small surface concentrations, then the adsorption isotherm, over a limited range of very small surface coverages, will be expected to be linear, irrespective of the nature of the sorbent surface (1). This undoubtedly accounts for the success of the GSC method when applied to volatile gas mixtures, using such adsorbents as silica gel, active charcoal, and zeolites. AS SOLID
The range of isotherm linearity may be considerably extended if the adsorbent surface is homogeneous. In this case, isotherm curvature will be associated with adsorbate-adsorbate interactions, or the filling of a significant proportion of the adsorption sites with adsorbed material. White and Cowan (12)attempted to obtain such a surface, by using an organo clay derivative as adsorbent, in which an aliphatic amine was base-exchanged on the clay. When the amine molecule chosen was sufficiently large, the surface could be covered with a homogeneous layer of adsorbed organic material. Eggertsen, Knight, and Groennings (4)obtained an effectiveIy homogeneous surface in a different Fay. They used a heterogeneously surfaced, pelleted, furnace, carbon black (Pelletex, Godfrey Cabot Co., Boston, Mass.) and then effectively removed the active sites by adsorbing a small amount of squalane on the surface. The effect of the squalane was demonstrated by comparing the chromatograms obtained from a mixture of 2:4 dimethyl pentane and cyclohexane on
