Detection of Polynuclear Hydrocarbons in Mixtures by Fluorescence Spectroscopy SIR: Fluorescence spectroscopy has been used for the identification of polynuclear hydrocarbons since the early studies of chemical carcinogenesis. The intense fluorescence of benzo(a)pyrene led to the isolation of this compound from coal tar (1). I n recent years, particularly since the introduction of improved fluorometers, much attention has been paid to the fluorescence of polynuclear hydrocarbons in the detection of these compounds (some of which are carcinogenic) in tobacco smoke (6, 6) and atmospheric pollutants (2). Instruments such as the Aminco and Farrand spectrofluorometers have been used to detect polynuclear compounds at concentrations much lower than those detectable by absorption spectroscopy. Sawicki, Hauser, and Stanley (3) and Van Duuren (4)have studied the fluorescence emission and excitation spectra of many polynuclear compounds and discussed their applkation to the identification of such compounds in mixtures. Although the fluorescence methods are mainly confirmatory of compounds identified by absorption spectroscopy (the components of the mixtures having Been separated by chromatography), in some cases--e.g., fluoranthene and pyrene ($)-reliance is placed on the fluorescence spectra to identify the components of mixtures not resolved by chromatography. Van Duuren (4) has discussed the difficulties encountered in resolving some mixtures by fluorescence spectroscopy. This investigation was undertaken to discover the limits of identification of the components of some commonly found and difficult to resolve mixtures. EXPERIMENTAL
An Aminco spcctrophotofluororneter with and l/lsinch slits was used in this study. The solvent was 2,2,4trimethylpentane (Phillips Petroleum Co.), chromatographed on silica gel (grade 922, Davison Chemical Co.) and distilled; it fluoresced only a t the highest sensitivity setting of the instrument. The polynuclear hydrocarbons were obtained from L. Light & Co., England. Standard solutions of the hydrocarbons containing 1 pg. per ml. were prepared and diluted appropriately for each experiment. RESULTS
The five mixtures studied were: pyrene plus fluoranthene, chrysene plus benz(a)anthracene, benzo(a)pyrene plus benzo(g,h,iiperylene, anthanthrcne plus benzolg,h,i)perylene, and anthanthrene plus benzo(a)pyrene. Both the 1448
ANALYTICAL CHEMISTRY
Table 1.
Mixture
Detectability of Components of Binary Mixtures
Concn., pg./Ml.
Identifiable FluoActivation res- Fluorescence Wave cence Wave Length, Spec- Length, Ratio Mp trum Mp
giz:&thene P rene FYuoranthene Pyrene Fluoranthene Pvrene Fhoranthene Pvrene Fhoranthene Pyrene Fluoranthene
0.5 0.5 0.25 0.5 0.1 0.5 0.5 0.1 0.5 0.05 0.5 0.01
1 330 1 360 1 330 2 360 1 330 5 360 5 330 1 360 10 330 1 360 50 330 1 360
Chrysene Benz(a)anthracene Chrysene Benz(a)anthracene Chrysene Benz(a)anthracene Chrysene Benz(a)anthracene
0.5 0.5 0.5 0.1 0.5 0.05 0.1 0.5
1 270 1 340
5 270 1 340 10 270 1 340 1 270 5 340
Benzo(a)pyrene Benzo(g,h,i)perylene Benzo(a)pyrene Benzo(g,h,i)perylene Denzo(a)pyrene Benzo(g,h,i)perylene Renzo(a)pyrene Benxo(g,h,i)perylcne
0.5 0.5 0.1 0.5 0.05 0.5 0.5 0.1
1 380 380 1 1 380 380 5 1 380 380 10 5 380 380 1
Anthanthrene Benzo(g,h,i)perylene Anthanthrene Benzo(g,h,i)perylene Anthanthrene Benzo(g,h,i)perylene Anthanthrene Benzo(g,h,i)perylene Anthanthrene Benzo(g,h,i)perylene Anthanthrene Benzo(g,h,i)perylene Anthanthrene Benzo(g,h,i)perylene
0.5 0.5 0.5 0.1 0.1 0.5 0.05 0.5 0.01 0.1 0.005 0.5 0.001 0.1
1 5 1 1 5 1 10 1 10 1 100 1 100
Anthanthrene Benzo(a)pyrene Anthanthrene Benzo(a)pyrene Anthanthrene Benzo(a )pyrene Anthanthrene Benzo(a )pyrene Anthanthrene Benzo(a)pyrene Anthanthrene Benzo(a)pyrene Anthsnthrene Benzo(a)pyrene
0.5 0.5 0.1 0.5 0.001 0.01 0.0005 0.01 0,001 0.05 0.0005 0.05 0.0005 0.1
1 430 1 360 1 430 380 5 1 400 380 10 1 400 20 380 1 400 50 380 1 400 380 100 1 400 380 200
+ + + + ++ ++ ++ +-
++ ++ + -
+ -+
390 460 390 460 390
460 390 460 390 460 390 370 420
370 420 370 420
370 420 410
+
410
-
410
-
+ + -
460
430
430 430 410 440 410
380
440
430 380
420
440
430
420
380 440
430
420
380
440
430 380
460
400 380
++ 4 4 0 ++ ++
++ ++ -
+ +
Activation spectra of two compounds indistinguishable
410
380 430
++ ++ ++ ++ ++ -+ ++ ++ ++ ++
430
440
1 430
Identifiable Activation Spectrum
420 420 410
440 410
460 460 460 460 460
410 410 410 410 410
++ -+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
relative and absolute concentrations of the components were varied and the effect on the erne of detection of each compound was studied. The fluorescence emission and activation spectra of each of the components of the mixtures were obtained by using the appropriate activation and emission wave lengths, respectively, for each compound. The results are shown in Table I. Fluoranthene could be identified even in the presence of a large preponderance of pyrene. Chrysenc and benz(a)anthracene could be ident,ified from the activation spectra, even with a large excess of one over the other. In the emission spectra benz(o)anthracene obscured chrysene in a 5 to 1 mixture. The activation spectra of benzo(a)pyrene and benzo(g,h,i)perylene were almost identical and could not be’ used to identify either in mixtures of the two. Benzo(g,h,i)perylene could not be detected by emission spectrum in the presence of benzo(a)pyrene unless the ratio of the former to the latter was 10 to 1 or greater. Anthanthrene and
benzo(g,h,i)perylene could be identified from the activation spectra, unless anthanthrene was in excess. Using emission spectra, benzo(g,k,i)perylene could not be detected in the presence of anthanthrene unless the concentration of the former was of the order of 100 times that of anthanthrene. I n the presence of benzo(a)pyrene anthanthrene could not be detected a t B ratio lower than 1 to 100 by activation spectra and 1 to 50 by emission spectra. It appeared that the relative eoncentration was of greater importance than absolute concentration in identifying the components of mixtures, particularly a t low concentrations. Difficulty was experienced in relating the fluorescence intensities to the concentrations of the components of mixtures. For example, anthanthrene a t the same concentration in the presence of different concentrations of benzo(a)pyrene gave fluorescence intensities of 42 and 66 units in the emission spectra, 19 and 6 units in the activation spectra. Similar variations were observed with
the other mixtures. It appears that quantification in fluorescence spectroscopy may be inaccurate in the presence of a second fluorescent compound. LITERATURE CITED
(1) Cook, J. W., Hewett, C. L., Hieger, I.,
J . Chem. SOC.1933,395. (2) Sawicki E Elbert, W T. w., .t-Iauihr, T. R., ANAL.CHEM.32,810 (19CO). (3) Sawicki, E., Hauser, T. R., Stanley, T. W., Intern. J . Air. Pollufion 2, 253 ( 1960). (4) Van Duuren, B. L., ANAL.CHEM.32, 1436 (19GO). (5) Van Duuren, 13. L., J. Nall. Cancer Inst. 21, l(1958). (6) Zbid., p. 623. WILLIAM C . LIJINSKY R. RAHA
F;x,SPIT:
ANNE CHESTNUT
Division of Oncology The Chicago Medical School 2020 West Ogden Ave. Chicago, Ill. INVESTIGATION supported hy grant (2-5170 from the National Institutes of Health, U. S. Public Health Service.
NOTE OF EXPLANATION
1,2,3-Tris(2-cyanoethoxy)propane, a Stationary Liquid for Gas Chromatography Columns; a Scientific Communication by H. M. McNair and Thomas De Vries, ANAL. CHEM. 33, 806
(1961) A manuscript entitled Cyanoethylated Glycerol m a Substrate for Gas Chromatography by E. S. English and E. M. Bevilscqua was rejected by ANALYTICALCHEMISTRY because Of insufficient originality. These authors had utiliaed the suggestion of H. M. Tenney [ANAL. CHEM. 30, 2 (1958)l in applying cyanoethylated glycerol as a substrate and also described its application in the investigation of the volatile products formed during the oxidation of rubber [ J . Org. Chem. 25, 1976 (1960)l. Bevilacqua and English have written to the editor, pointing out that the earlier publications anticipated
the Scientific Communication of McNair and De Vries, as well as their own unpublished article. Unfortunately, neither the editor nor reviewers of the McNair-De Vries article noted the similarity in general subject matter between this article and the publications of Tenney and of Bevilacqua and English. However, correspondence between the editor and the two groups of authors has brought to light that, in addition to the above work, mention of the use of the substrate was also made by:
A. C. Cope and P. E. Peterson, f o o t
note to their article in J . Am. Chern. SOC.81, 1647 (1959). B. C. Anderson a t the Second International Symposium on Gas Chromatography, June 1959; paper now included in “Gas Chromatography,” Academic Press, Inc., 1961. I n their Scientific Communication, McNair and De Vries did not claim priority but showed the extreme selectivity of the substrate and its use to a t least 153’ C. These comments are offered as a matter of record to correct the literature.
VOL. 33, NO. 10, SEPTEMBER 1961
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