LITERATURE CITED
(1) Brochmann-Hanssen, E., J . Pharm.
S U . 51, 1017-31 (1962). (2) de Silva, J. A. F., D'Arconte, L., Kaplan, J ., Current Therapeutzc Research 6 , 116-21 (1964). ( 3 ) Homing, E. C., Moscatelli, E. A , , Sweeley, C. C., Chtam. Ind. (London) 1959, 761-2. (4)Horning, E. C., Vanden Heuvel,
W.J. A., Ann. Rev. Biochem. 32, 709-54 (1963). ( 5 ) Koechlin, B. A., D'hrconte, L., Anal. Biochem. 5,195-207 (1963). (6) Koechlin, B. A , , Oberhansli, W., D'A4rconte,L., Dept. of Pharmacology, Hoffmann-La Roche Inc., Sutley, N. J., unpublished data, June 23, 1963. (7) Randall, L. O., Dzseases Servous System 22, Suppl. 7 , 1-9 (1961). (8) Randall, L. O., Heise, G. A., Srhallek, W., Bagdon, R. E., Banziger, R., Boris, A., Moe, R. A., Abrams, W. B.,
Current Therapeutic Research 3, 405-25 (1961). (9) Schwartz, M. .4.,Koechlin, B. A., Krol, G., Federation Proc. 22, Part 1, No. 2, 367 (1963). (IO) Sternbach, 1,. H., Fryer, R. I., bletlesics, W., Sach. G., Stempel, A . , J . Org. Chem. 27, 3781-8 (1962). (111 Sternbach. L. H.. Reeder., E.., Ibid.. 26,4936-41 (1961). '
RECEIVEDfor review May 14, 1964. Accepted July 21, 1964.
Analysis of Aromatic Hydrocarbons from Pitch Oils by Liquid Chromatography on Gas Chromatolg ra phy An a Iog CLARENCE KARR, Jr., EDWARD E. CHILDERS, WILLIAM C. WARNER, and PATRICIA E. ESTEP Morgantown Coal Research Center, Bureau o f Mines,
b Liquid chromatography on the gas chromatography analog has been demonstrated for the first time to b e an effective method o f analyzing complex natural mixtures of high boiling aromatic hydrocarbons, including the identification of individual constituents. Pitch oil fractions boiling in the range 290" to 315" C. were analyzed b y this method and shown to contain about 70 components. O f these 1 1 were readily identified b y relative retentions and b y infrared and ultraviolet spectra. Those identified were dibenzofuran, fluorene, their various methyl derivatives, and phenanthrene. Many additional compounds h e r e partially or tentatively identified. The method also proved to be convenient for purifying samples of compounds and detlermining their impurities.
N
PREVIOUS STUDIES have been reported on the separation of complex natural mixtures of high boiling aromatic hydrocarbons by liquid chromatography on the gas chromatography analog, and the identification thereby of individual constituents. This analog (3) requires a column with a relatively large length-to-diameter ratio (typically obtained in gas chromatography with 'j4-inch metal tubing); a column packing that can be used for many runs without regeneration; a single carrier fluid that is used throughout a run, such as helium in gas rhromatography; automatic recording of the concentration peaks in a chromatogram; and, ideally, reproducible retention volumes for the chromatographic peakE, as a n aid in identification of components. For a complete analogy, the efficiency of the column should approach t h a t of a gas
o
U. S.
Department o f the Interior, Morgantown, W. Va.
chromatographic column, but this is impractical ( 2 ) . -4pparently the only previous instance in which a complex natural mixture of polycyclic aromatic hydrocarbons was separated by liquid chromatography and the constituents identified is the work on atmospheric pollutants by Cleary ( 1 ) . In this study, a 'I2X 23inch column of alumina containing 13 to 13.5 weight yo water was used witli cyclohexane as a first eluent, followed by cyclohexane with gradually increasing concentrations of ethyl ether. No chromatograms were recorded, and although qualitative orders of elution were determined, retention volumes were not. In the present work, liquid chromatography on the gas chromatography analog (3) was used to analyze pitch oil fractions which boil a t 290" to 315" C. from a low temperature bituminous coal tar. This method was also convenient for the purification of compound samples and the dermination of their impurities. EXPERIMENTAL
The column was a %-foot length of 1/4-inch copper tubing packed with 162.95 grams of 80- to 100-mesh activated F-20 alumina containing 4.09 ' water. The carrier fluid was weight % spectral grade cyclohexane a t 125 p.s.i.g. and room temperature. Chromatograms were automatically recorded with one of the commercially available ultraviolet absorption monitoring devices. The dual-pen recorder plotted absorbance us. effluent volume; the marginal pen indicated each 400drop fraction collected in an automatic, drop-counting fraction collector. .it room temperature there was approximately 11.5 ml. of cyclohexane in each fraction. Chromatograms obtained with this device had the advantage of
built-in attenuation insofar as the recording of absorbance was a logarithmic function and could only approach the top of the chart, never reach it. However, this device has a disadvantage to those who are familiar with the conventional gas chromatograms because it greatly deemphasizes the valleys so that only a slight overlap of highly absorbing compounds could result in two greatly overlapping peaks on the chromatogram. The pitch oil fractions were obtained by distillation in a spinning band column with a head pressure of about 0.015 mm. of Hg. An accurate determination of the equivalent atmospheric boiling points was impossible to make, but these appeared to be in the range 290" to 315" C. for the six consecutive fractions examined. The charge to the column was 20 to 25 mg. of pitch oil and 20 p1. of o-cthyltoluene (OET) as the internal standard. Samples were introduced through a T-fitting in the line just preceding the packing. Because no device was available to inject liquids against a pressure of 125 p.s.i.g., the following technique was used. The line to the pressurized tank of cyclohexane was closed and the column exit opened to allow the pressure in the column to fall to atmospheric. The cap on the T-fitting was removed and a little more cyclohexane drained from the column by gravity flow until only a thin layer covered the top of the bed. T h e charge was then carefully deposited by a syringe and needle onto the top of the bed and allowed to flow down to form a thin layer again. This last operation was repeated for a cyclohexane rinse. The column exit was closrd, the T-fitting completely filled with cyclohexane so as to displace all air, and the cap secured. Finally, the line to the pressurized tank was opened, then the line to the column exit. Conducted in this manner, no disturbance of the packing was ever detected. VOL. 36, NO. 11, OCTOBER 1964
2105
Each complete run, with the collection of about 60 fractions, took about 7 hours. The runs could have been completed faster because the system could have been operated up to the 300-p.s.i.g. rating of the safety rupture disk, but the resolution of components was not as good a t these faster flow rates. Water content and particle size of the adsorbent also affected the degree of separation. Lower water content gave better separations but greatly increased retention volume. Smaller particle size gave better separation but required a much higher operating pressure for a practical flow rate. Increased column length gave better separations but proportionately decreased flow rate a t the same pressure. With these various factors working in opposition, the combination obtaining the best separations within the one workday time limit was not readily determinable. Giddings (2) has determined from purely theoretical considerations that the optimum conditions would be centered on a n extremely small particle size of the adsorbent. However, there is no evidence that this has been demonstrated in the laboratory. Many aromatic hydrocarbon compounds were run both to obtain relative retentions and to determine the impurities present in these samples. In all runs on pitch oils and compound samples, the ultraviolet spectra were conveniently obtained on the cyclohexane solutions from tubes corresponding to the chromatogram peaks. The quantitative isolation of components presented none of the problems that occur when gas chromatography is used. The infrared spectra a e r e obtained in carbon disulfide with ultramicrocavity cells and a beam condenser after the cyclohexane was evaporated with a stream of nitrogen from a syringe needle. AI1 of the work reported here and many additional tests were performed with this same column without changing the packing, without using a carrier fluid other than cyclohexane, and without subjecting the packing to any treatment. The performance of the column appeared to remain unaltered throughout all this work for the retention volume of the O E T remained regular.
Table I.
NO.
Constituent Identity
FRACTION So. 2
2 106
ANALYTICAL CHEMISTRY
11.4
1 Internal standard,
2 4 5 6
o-ethyltoluene (OET) Alkylbip henyl Unknown I Unknown I1 Alkyl 1,2,3,4-tetrahydrophenanthrenes
7 Dimethylbiphenyls Trimethylnaphthalenes 9 10 11
12 13
N1 Nz NI Sr
x,
Trimethylnap hthalenes
14 Xe 15 N 7 16 N 8 25 Unknown V 17 Alkyldiphenyl ether 19 4-Methyldibenaofuran
18 Dibenzofuran
26 9-Methylfluorene 27 Unknown VI 20 2-Methyldibenzofuran
21
1-Methyldibenzofuran
22 3-Methyldibenaofuran
RESULTS A N D DISCUSSION
Four chromatograms typical of those observed during the purification of aromatic hydrocarbon compounds are shown in Figure 1. The amount of charge varied from about 8 to 16 mg., except for acenaphthylene which was 106 mg. In all cases, 20 pl. of O E T was added so that relative retentions could be obtained. The six chromatograms for the six consecutive pitch oil distillate fractions are -horn in Figure 2. The detailed results for two of these six fractions are given in Table I. These qix chromatograms demonqtrate that this technique readily shows changes in composition
0bserved analytical wavelengths (UV in mp, I R in p )
Liquid Chromatography Peak tube No. Amt. of ChroconmatoSpecstituent graphic tral
23 Fluorene
28 3-Methylfluorene 29 2-Methylfluorene
30 Unknown VI1 31 Cnknown VI11
250-260 mp (broad) 226 mp 260 mp 321, 314, 290, 283, 278, 273, 228.2, 223 mp 13 44, 12 36, 9 68 p 250-260 mp (broad) 14 37, 13 47, 13 30, 12 97, 12 40, 11 45 p 324, 317, 310, 280, 231, 225 mp 13 44 p (3 or 4 free H ) 12 57 p (2 free H) 12 40 p (2 free H) 12 15 p (2 free H ) 11 70 p (1 or 2 free H ) 323, 317 5 , 308, 280, 230 2, 225 mp 12 35 p 12 65 p 12 0 3 p 13 5 O p 14 50, 8 08 p 302 5 , 296 1, 291, 285.5, 280 4, 272, 252.5, 246.3, 243 2 mu 13.72, i 3 32, 12.75, 12.05, 11.83, 9.16, 8.42 p 302, 296 5, 285 7, 280.2, 275 2, 248 8, 243.7, 241 mp 10.80, 9.77, 9.09, 9.01, 8.38, 8.05, 7 . 8 0 ~ 13 58, 13 19 1.1 338 mp 307 5, 295, 285 5, 251, 243 mp 13 87, 13 42, 11 90, 11 47, 9 70, 8 93 8 38, 8 03, 7 81 p 303, 293 5, 280, 253, 245 mp 13 83, 10 50, 9 30, 8 14p 307 5 , 298, 289, 284, 251, 246,243 mp 13 84, 13 38, 9 82, 8 88, 8 32, 7 92 p 299 5,292 3,288 3,277, 270 6, 260 4, 254 5 , 226, 219 6 mp 14 46. 13 56. 10 50. 9,97, 8.43,’7.72, ’ 7.15 p 304, 296 7, 292, 262 mp 13.73, 13.07,12.45, 8.60 p 304.5, 298, 293, 280.2, 276, 271, 265, 258, 227 7, 223 mp 13 72, 13 12, 12 22, 10 5 , 7 68 p 344, 337, 328, 251 mp 12 45 p
Minor Minor Minor Major
15.5 17
Major
13-14 14 14-15 16-17 17
19.6 Minor Minor Minor Minor Major Major Major Minor Trace Trace Major
21.8
24.5
19 19 19 19 20 21 21 21 21 24 24
Minor
25
Minor Trace Major
25 25-26 27
28 2
Minor
27
Major
29
Major
30 3
30
Major
35.9
34
Major
36
Trace Minor
31-32 33-34
of Pitch Oil Fractions
-
.-
NO.
Constituent Identity
Observed analytical wavelengths (UV in mp, I R in p )
Amt. of constituent
Table II. Comparison of Relative Retentions; Effect of Natural Mixtures
Peak tube No. -
Chromatographic Spectral
FRACTION KO.5 1 Internal standard,
o-ethyltoluene 1 OETI 7 Polyalkylbip heny 1s 250-260 mp (broad) 14 37, 13 47, 13 30, 12 97, 12 40, 11 45 p 42 3-Ring hydroaromatic 270, 228 mp TrimethyInaphth,&nes 323, 317 5, 308, 280, 230 2, 225 mp 14 Ng 12 3 5 p 12 0 3 u 16 Ns Polyalkylnaphthalenes Tg 43 327, 280, 231.7 mp 12.70 p 44 12 15 p Nlo 45 323, 231 7 mp, 12 80 p N1I 46 Dimethyldibenzofuran 308, 29.5, 283, 251 4 mp 13 42, 13 06, 12 38, I 8 43 p 47 Diniethyldibenzofuran 252 8 mp 11 48 Dimethyldibenzofuran 307, 298 5, 290-280, 250 8 mp 13 85. 13 40. 13 10. 12 32, 11 63, 11 42, 10 98, 10 80, 9 80, 9 70, 9 47, 9 08 8 92, 8 36, 8 16, 8 03. 7 83. 7 56 u Tetramethylnaph327 8, 321.5; 290-280, thalene 234 mp 49 12.32 p ( 2 free H ) XI2 50 2,4-Dimethyldibeiizo309, 297, 286, 283, 253 6, furan 245 mp 13 75, 13 39, 13 08, 12 46, 12 02, 11 77, 9.88, 9 67, 9 15, 8 92, 8 73, 8 44, 7 92, 7 62p 21 1-Methyldibenzofuran 303, 293.5, 280, 253, 245 mp 13 83,10.50,9.30,81.4p 51 1,3-Dimethyldibenzo9.52 p furan Tetramethylnapht ha- 329.8, 322.5, 290-280, 233.5 mp lene 52 13 42 p El8 53 Dimethyldibenzof uran 305, 298, 289 2, 253 5, VI 245 mp 8 40p 54 Diniethyldibenzof man 287, 253 5 mp VI1 12 45, 8 30 p 55 Unknown XI1 224 m p 56 Unknown XI11 253 mp, 12 45 p 28 3-hf ethylfluorene 304, 296.7, 292, 262 mp 13.73,13.07,12.45, 8.60 p 29 2-llethylfluorene 304.5, 298, 293, 280.2, 276. 271. 265. 258. ~, 227'.7, 223 m; 13.72,13.12,12.22,10.5, 7.68p 301 2, 295, 289 5, 178.4, 36 1-Methylfluorene 273,266 3,262,256 4 14m15, 13 77, 13 30, 12 66, 11 65, 10 59, 9 78p 57 Carbonyl compound IV 261 mpr 5,84, 5 . 7 5 p
11 0
Minor
17.4
Minor 20 Major Minor 23.1
17 17 21-22 20
Minor
21-22
Minor Minor Major
23 23 24-25
24
Trace Major
26 28
Major
Major
28
29
31.9
31
Minor
31
Trace
33
Major
35
Major Major Minor Minor Minor
39 41
41-42
44
41 42 44
Minor
Minor
Minor
35
48
52
52
5
Relative retention" Pitch Pure 011 comcomCompound pound ponent* Dibenzofuran 2 2 2 3 4-Methyldibenzofuran 2 3 2 4 2-Methyldibenzofuran 2 5 2 7 3-Methyldibenzofuran 2 7 2 8 Fluorene 3 1 3 1 Phenanthrene 3 7 3 7 3-Methylfluorene 3 8 2-Methylfluorene 4 4 4 3 1-Methylfluorene 4 9 4 7
I
a
Relative t o o-ethyltoluene. Average of all runs where found
from one narrow boiling fraction to the nest throughout a series of fractions. The numbers on the chromatographic peaks refer to some of the constituents listed in Table 1. These chromatograms can be misleading because the recording of a logarithmic function greatly emphasizes the overlap between peaks t h a t , on a linear scale, would appear negligible. The actual degree of separation of components was discovered upon examining the ultraviolet and infrared spectra of the contents of successive tubes. In many instances, the spectra for adjacent tubes changed so much as to indicate only a 5 or 10 mole % overlap of components, and in some instances the spectra of isolated components were better than those of socalled pure samples as received. This situation can be realized from the spectral d a t a in Table I. There were 11 major components of these pitch oil fractions that were readily identified by their spectra and relative retentions. These were dibenzofuran, its 1-, 2-, 3-, and 4-nlethyl and 2, 4-dimethyl derivatives; fluorine, its I-, 2-, and 3-methyl derivatives; and phenanthrene. An additional 40 to 45 components were partially or tentatively identified, and the presence of a t least 15 completely unknown constituents was determined, making a total of about 70 compounds indicated as being present in pitch oil covering only a 25" C. boiling range. Although the relative retentions of the various compounds always remained in the same order, their numerical values as determined from these complex pitch oils frequently shifted a little to lomer or higher values compared with the compounds run by themselves with the internal standard. This effect is summarized in Table I1 for nine of the constituents Studies with synthetic mixtures indicated that these discrepancies a e r e VOL. 36, NO. 11, OCTOBER 1964
2107
FRACTION 4
FRACTION I f7
20 23 I
I
I
I
I
1
IO
20
30
40
I
I
I
I
I
I
I
I
50
I
IO
20
30
40
50
60
FRACTION 2
FRACTION 5
d I
I
I
10
I
I
I
I
20
30
40
50
I
I
I
(
IO
1
I
I
10
20
30
I
I
10 TUBE N U M B E R
I
I
I
I
20
30
40
50
Figure 1 . Purification of aromatic compounds by liquid chromatography
I
I
I
IO
~
20
I
30
(I
o-ethyltoluene, internal standard; ( 2 ) acenaphthene, impurity; ( 3 ) ocenaphthylene; (4) dibenzofuran; (5) impurity; (6) 1,2,3,4-tetrohydro9,lO-dihydrophenanthrene; ( 8 ) phenanthrene, phenanthrene, impurity; (7) impurity; ( 9 ) 9-methylfluorene; ( I 0)fluorene, impurity
caused by the amount of the compound and by the presence and nature of other compounds with similar retention volumes. Relative retentions are thus best determined from synthetic mixtures similar both qualitatively and quantitatively to the sample being analyzed. Relative orders of elution of constituents remained invariant and in actual practice these orders were adequate for tentative identification, especially when examining a series of fractions.
I
40
I
30
40
I
50
I
60
FRACTION 6
FRACTION 3
-
1
20
I
I
~
1 10 T U B E NUMBER
50
I
20
I
30
I
40
I
50
60
Figure 2. Liquid chromatography of consecutive pitch oil distillate fractions Numbers on chromatographic peaks refer to some of the constituents listed in Table I
ACKNOWLEDGMENT
The authors thank Chester M u t h , Chemistry Department, West Virginia University, for a sample of 4-methyldibenzofuran; . I . Ladam, Centre des Gtudes et des Recherches des Charbonnages de France, for samples of 1-, 2-, and 3-methylfluorene and 2- and 3methyldibenzofuran, and S. Trippett, University of Leeds, England, for
spectra of a benzofurans.
variety
of
alkyldi-
LITERATURE CITED
(1) Cleary, G. J., J . Chromatog. 9, 204 (1962). (2) Giddings, J. Calvin, ANAL.CHEY.35,
2215 (1963).
( 3 ) Ilarr, C., Jr., Childers, E. E., \Tamer) W .C., Ibid., p. 1290. RECEIVEDfor review April 29, 1964. Accepted July 28, 1964.
Separation and Analysis of Alkylpolynitrates by Gas Chromatography ETTORE CAMERA and DARIO PRAVlSANl Dinarnite, S.p.A., Mereto di Tornba, Udine, Italy'
b Monoethylene glycol dinitrate, glycerol trinitrate, diethylene glycol dinitrate, triethylene glycol dinitrate, 1,2-dinitropropanediol, and 1 ,5-dinitropentanediol were separated by gas liquid chromatography. Ethylene glycol succinate was used as the partition liquid, and Celite C 22 ak was used as support. Decomposition of glycerol trinitrate was minimized by very short columns and the temperature was kept as low as possible. Quantitative data are given, and maximum deviations from the mean that average iO.0423 show that the method is precise. 2 108
ANALYTICAL CHEMISTRY
N
were separated and analyzed by distillation and crystallization by Marqueyrol and Goutal (S), and a solvent extraction method was developed by o h m a n (4,5). ,\mong the esters studied by Bhman were glycerol trinitrate (NG), ethylene glycol dinitrate (EGDN), and diethylene glycol dinitrate (DEGN). Pristera et al. (8) and Carol ( 1 ) used an infrared method to analyze mixtures of nitric esters and other explosive compounds. Chromatographic methods were studied by Schroeder (10) and Ovenston ITRIC ESTERS
(6). Pollard and Hardy analyzed simple alkylnitrates by gas chromatography (?), and Evered and Pollard obtained retention data and activity coefficients for some nitric esters ( 2 ) . So far as we know, no quantitative analysis of alkylpolynitrate esters by gas chromatography has appeared in the literature. This is no doubt due, in some part, to the decomposition of N G which begins at about 80" C. and increases rapidly with increasing temAffiliated with Research Dept. of Dr. Ing. M, Biazzi Soc. An., Vevey, Switzer-
land.
I
