the standard solutions are - 10% of the initial values. Thus it is evident that the colors are stable enough for analytical work. The absorption spectrum of the blank as compared to 10% aqueous dimethylformamide is practically negligibleabsorbance of 0.1 a t 582 mp. Reproducibility in absorbance obtained with standard formaldehyde solutions is excellent. For example, over a period of a month 20 determinations of solutions containing 50 pg. of formaldehyde gave absorbance values of 0.79 i 0.02. Beer’s law also was found to hold over a range of 10 to 150 pg. per 100 ml. of final solution. Variables in the colorimetric procedure were investigated. An increase in the concentration of the hydrazinobenzothiazole caused an increase in the color of the blank; a decrease in concentration caused a decrease in the intensity of the blue color in the standard solution. At least 5 minutes’ standing was necessary for the formation of the hydrazone; a shorter reaction time caused a decrease in the intensity of the blue color. The smaller the concentration of potassium ferricyanide, the longer the oxidation period to obtain maximum color development. Using a 2% solution gave good results, but a bluish colored blank. A 3y0 solution of potassium ferricyanide produced a precipitate. The most satisfactory results were obtained with the 1% solution and a 17- to 25-minute oxidation
period. For example, in a final solution containing 100 pg. of formaldehyde, oxidation times of 5, 10, 15, 20, 25, and 30 minutes gave absorbance values of 1 .OO, 1.25, 1.57, 1.59, 1.58, and 1.56, respectively. Heat applied durmg the oxidation step in an attempt to shorten the time resulted in decomposition. The reddish brown precipitate of the formazan obtained after the oxidation step is so insoluble that formaldehyde could probably be determined gravimetrically by this method. This precipitate can also be extracted with chloroform or o-dichlorobenzene and then made alkaline t o give a purple (chloroform) to blue (0-dichlorobenzene) color. Once the blue dye mas formed, some dimethylformamide was necessary to keep it in solution. The presence of 10% dimethylformamide gave satisfactory results. A somewhat greater concentration gave a more highly colored blank. With a further increase in concentration of dimethylformamide, the inorganic salts tended to precipitate. The concentration of the alkali was not too critical. Doubling the alkali concentration had no effect on the color intensity. The procedure of Wilson (7) was used for preparing and collecting air-aldehyde mixtures for analysis by the impinger method. However, in 7itTilson’s method the gas is collected in aqueous sodium bisulfite, while in the present method 0.25% 2-hydrazinobenzothiazole solution is used. One hundred and ninety-
eight micrograms of formaldehyde were driven over into 20 ml. of the collecting solution six times, with analyses of 10-ml. portions each time. Absorbance values of 1.56 0.02 were obtained. Because a theoretical absorbance value of 1.59 should have been obtained, efficiency in the collection of aldehyde is in the neighborhood of 97 to 99%. Some interferences were noted. Nitromethane gives a blue color in very high concentrations and an absorbance of about 10. The larger aliphatic aldehydes were somewhat water-insoluble and gave essentially negative results in the colorimetric procedure.
*
LITERATURE CITED
(1) Fe,ig!! F., “Spot Tests in Organic Analym, 5th ed., p. 348, Elsevier, New
York, 1956. (2) Kersey, R . W., hladdocks, J. R., Johnson, T. E., Analyst 65, 203 (1940). (3) Sanicki, E., Stanley, T. W., Hauser, T. R., ANAL.CHEM.31, 2063 (1959). (4) Schryver, S. B., Proc. Roy. SOC. (London) 82B, 226 (1910). (5) Tanenbaum, hl., Bricker, C. E., ANAL. CHEM.23, 354 (1951). (6) Thomas, J. F., Sanborne, E. N., Mukoi, M., Tebbens, B. O., A . M . A . Arch. Znd. Health 20, 420 (1959). ( 7 ) \viISOn, K. Nr., A N A L . CHEM. 30, 1127 (1958). (8) Yoe, J. A,, Reid, L. C., IND.ENCI. CHEM.,ANAL.ED., 13, 238 (1941). (9) Zurlo, N., Griffini, A. M., M e d . laooro 45, 692 (1964). RECEIVED for review February 15, 1960. Accepted June 8, 1960. Air Pollution S mposium, Division of Water and Waste &emistry, 138th Meeting, ACS, New York, N. Y., September 1960.
The Fluorescence Spectra of Aromatic Hydrocarbons and HeterocycIic Aromatic Compounds BENJAMIN 1. VAN DUUREN Institute o f lndustrial Medicine, New York Universily Medical Center, New York, N. Y.
b The application of fluorescence spectroscopy in the qualitative and quantitative analysis of aromatic compounds was investigated. The replacement of hydrogen atoms of aromatic hydrocarbons by alkyl groups in some instances gives noticeable shifts in fluorescence spectra. The replacement of carbon atoms of aromatic hydrocarbons by nitrogen and by oxygen was also examined. Certain compounds show markedly different fluorescence behavior in different solvents or in their ionized forms. This relationship between changes in fluorescence spectra with changes in solvents was compared with the effects of the same solvents on the ultraviolet absorption spectra. Fluorescence excita1436
e
ANALYTICAL CHEMISTRY
tion spectra obtained with an automatic recording spectrofluorometer augment identifications of compounds made on the basis of ultraviolet and fluorescence emission spectra. The effect of concentration on fluorescence intensity was also examined.
R
in instrumentation and the availability of commercial automatic recording spectrofluorometers have made possible the more extensive use of fluorescence spectroscopy for analytical purposes. It has served as a valuable tool in this laboratory in the identification of cigarette tar aromatic hydrocarbons (13, 14) and heterocyclics (16). The aromatic compounds were separated ECENT ADVAKCES
and identified by their R , values, ultraviolet spectra, and fluorescence spectra. In the course of the author’s work on the cigarette tar aromatics, a number of aromatic compounds were purified and their fluorescence spectra examined. Some of the observations made, new data, and conclusions on the fluorescence of these compounds are described in this report. EXPERIMENTAL
of Aromatic Compounds, All compounds used in this Purification
work were subjected t o purification procedures, including column chromatography, crystallization, and/or vacuum sublimation. The criteria of
Table 1.
Relationship between Fluorescence Intensity and Concentration for
Band Maxima Sensitivity Concn., y / d . 0.3 0.1 0.2
0.1
0.15
0.1 0.01
0.10
0.04 0.02 0.01 Ult,raviolet maxima
0.01 0.01 0.01
292 2.8 1.6 0.7 0.47 0.19
0.11
0.06 286
Ultraviolet maxima, solvent benzene ( 8 )
290
Band Excitation (Emission at 400 Mp) 299 324 338 351 4.4 1.4 1.4 1.2 1.8 0.5 0.5 0.5 1.1 0.3 0.25 0.25 0.74 0.17 0.16 0.14 0.27 0.09 0.07 0.07 0.19 0.07 0.07 0.11 0.10 0.04 0.04 0.02 297 323 336 351 363
Emission maxima (10 ) a Arbitrary chart scale: for sensitivity 1.0, scale
purity used were melting points, ultraviolet absorption spectra, and the absence of other fluorescent compounds as shown by paper chromatography. Solvent. Except where stated
322
300
=
336
351
Intensity,
Dibenz[ a,h]anthracene
Mpa
Emission (Excitation a t 300 Mp) 406 420 440 454 1.8 2.7 0.9 0.1 0.7 1.0 0.4 ... 1.1 0 5 0.7 0.3 ... 0.75 0.35 0.53 0.17 ... 0.25 0.12 0.17 0.07 .. 0.19 0.09 0.13 0.05 ... 0.08 0.045 0.055 0.03 ...
375
397 3.9 1.4
0.1
... ... ... ... ... ... 373 382 392 373 385 395
394
406
416
444
.
0 to 100; sensitivity 0.1, scale = 0 to 10; sensitivity 0.01, scale = 0 to 1.
Table It.
Effect of Solvent on Fluorescence of Dibenzacridines
Band Intensity, Mp Solvent
Emission m a x i m a DIBENZ [U,h] ACRIDINE
Cyclohexane Sulfuric acid
294,319,332,353,374,384,393 220, 240, 263, 287, 295, 317, 332, 345, 353, 363,371,382,392 a 310, 400, 430, 442, 457 0 260, 300, 310, 417, 440 a
395,b 403, 418, - 445, 470 460, 485, 500 -
DIBENZ [a,j)ACRIDINE
395, 400,.-416, 440, 465 300, 321, 337, 355, 375, 393 223, 254, 283, 292, 299, 318, 333, 352, 362, 370,382,393 452, 478 Sulfuric acid 300, 425, 445 228, 290, 300, 410, 433 a Excitation maxima. b In Tables I1 to VI principal bands in emission spectra are underlined. c Ultraviolet absorption. Cyclohexane
>
-rn
I-
a
z W
I-
z W
0 2 W 0 v)
W
a
0 3 J
LL
250
300
MY
EXCITATION
350
400
SPECTRA
Figure 1. Fluorescence excitation spectra of 6-methylbenz [a]anthracene I . 10.0 y/d, (lens. 1.O) 2. 1 .O y/mt. (sens. 0.1 0) 3. 0.05 y/rnl.(sens. 0.01)
otherwise, the solvent used for all fluorescence and ultraviolet absorption spectra was spectroscopically pure cyclohexane (Matheson, Coleman and Bell). This solvent usually showed very little fluorescence even with the instrument a t its maximum sensitivity setting. Since most of the observations were made for qualitative purposes, oxygen was not rigorously excluded from the solvent. The concentrations of the aromatic compounds varied between 0.01 and 1.0 y per ml. Instrumentation. T h e Farrand automatic recording spectrofluorometer equipped with a high intensity 150-watt Hanovia xenon arc was used. The sample can be irradiated at a n y desired wave length between 220 and 650 mp through a grating monochromator. The measuring monochromator can be used a t any chosen wave length in the same range. Except where stated otherwise, 5-mp slits were used. A lP28 multiplier phototube Kas used for all fluorescence measurements. A fluorescence macrocell (10 X 20 X 50 mm.), requiring approximately 7 ml. of solution, was used in most of the work. A microcell (3 X 3 X 40 mm.) requiring only 0.3 ml. was used in some instances. Ultraviolet absorption spec-
tra were obtained with a Beckman DU spectrophotometer equipped with a Process and Instruments Co. automatic recording unit. RESULTS AND DISCUSSION
Calibration, Fluorescence Intensity, a n d Concentration. Quinine sulfate was used t o calibrate t h e spectrofluorometer, T h e excitation maximum is at 350 mp and the emission maximum (11 ) a t 450 mp. Sprince and Rowley (11) report a linear relationship between concentration and fluorescence intensity from 0.005 to 0.1 y per ml. of quinine sulfate in aqueous sulfuric acid, This agrees well with our measurements with the Farrand instrument. T o establish the most desirable range of concentrations in which to measure fluorescence spectra of the aromatic compounds, concentration-fluorescence intensity correlations were carried out for a number of these compounds of differing fluorescent properties. As a n example, the results obtained with dibenz [a,h]anthraceneare shown in Table 1.
Concentration bears a linear relationVOL. 32, NO. 1 1 , OCTOBER 1960
* 1437
>
c, tn 2 W
c-
E W 0 2 W 0
tn
240
W
260
2 so
30 0
L
320
340
360
38 0
400
MP
0
3
Figure 3.
J L
Spectra of dibenzo [a,i]pyrene I. 2.
i'
i i
i
I
I
,
350
400
450
EMISSION
'-..
MY
SPECTRA
Figure 2. Fluorescence emission spectra of 6-methylbenz [alanthracene 7. 2. 3.
10.0 ?/mi. (sens. 1.0) 1 .O y / d . kens. 0.1 0) 0.05 y/tnI. (sens. 0.01 )
Ultraviolet absorption Fluorescence excitation
ship to fluorescence intensity between 0.01 and 0.1 y per ml. for dibenz[a,h]anthracene within the limits of accuracy of the instrument. At higher concentrationa, the deviation from linearity becomes larger. The linear response occum also in the excitation spectra. I n the presence of traces of fluorescent impurities, the relative band intensities undergo changes. This phenomenon has been observed in the aromatic compounds obtained from cigarette tars (14, 16). The limits of accuracy in these quantitative analyses are on the order of *5%. The lower concentration shown in Table I, 0.01 y per ml., was also the lower limit for obtaining recognizable spectra of dibenz [a,h]anthracene with 5-mp slits. Dibenzo [a,i]pyrene, on the other hand, could be detected and acceptable spectra
Table 111. Fluorescence and Ultraviolet Absorption Maxima of Acridine Solvent Band Intensity, Mp Emission 400 a 248,325,339,347,355,368, . . . Cyclohexane 250, . . . , . . ., . . ,, . . ., 370, . . . 415,440 Ethyl alcohol a 248,325,339,347,355, . , . , 380 250, , . ., 340, . . ., 355, . . . ,380 430, 4 248, . . , , 338,347,355, , . , ,382 Water - 455, 475 * 250, . . ., . . ., . . ., 355, , . ., 382 457,484,505,515 254, . . . ,338,346,354, , . . , . . . Sulfuric acid (10% aq. and concd.) b 255, . , , , . , . , , . . , 355, . . . , . . . , 392, 403, 0
415,430,440
a
*
Ultraviolet absorption. Fluorescence excitation.
1438
ANALYTICAL CHEMISTRY
obtained at a concentration of only 0.001 y per ml. Some changes were observed in the relative intensity of the bands in the excitation and emission spectra with change in concentration. Short wave length maxima in both the, excitation and emission spectra became relatively more intense as the concentration of the solution was decreased (Figures 1 and 2). The fluorescence spectra of a numper of compounds obtained at different concentrations exhibited the same phenomenon. Foreter (5) observed similar changes in the fluorescence spectra of pyrene with change in concentration and ascribed i t to the formation of dimers between excited and unexcited molecules. The phenomenon is probably closely related to concentration quenching. Fluorescence Excitation a n d Ultraviolet Absorption Maxima. Theoretically, the fluorescence excitation maxima correspond in their positions with the ultraviolet absorption maxima. It was important to determine how close this agreement would be since two instruments are involved. This comparison was routinely carried out for all the compounds examined in this study. The actual appearance of such spectra is shown in Figure 3. From the evidence obtained, there is close agreement in these m v e lengths. The deviation in maxima is usually 1 3 mp, which is also the l h ' c of
x
rr)
d
I
9
250 300 350 W A V E L E N G T H , M)J
Figure 4. Fluorescence excitation spectra of indole a t
5.5 y/rnI. I. 2. 3.
In water In cyclohexane In ethyl alcohol
accuracy of measurements on both the instruments used. It is therefore possible to read some of the ultraviolet absorption maxima from the fluorescence excitation spectrum of a fluorescent compound. This procedure is particularly useful in those instances where trace amounts of products are isolated from complex mixtures. Thus, in the analysis of cigarette tars, background absorption sometimes obscures some of the ultraviolet absorption maxima (16). Acceptable fluorescence excitation spectra aid in qualitative identification in these cases. Analysis of Mixtures. The fluorescence exhibited by some compounds is so intense as t o be distinguishable even in very complex mixtures. This fact has been known for some time and was, in fact, used by Cook and coworkers (3) in the identification and isolation of benzo [alpyrene from coal tar. However, the detection of compounds with a lower fluorescence efficiency than benzo [alpyrene waB examined and found difficult. A mixture containing 1 y of each of anthracene, pyrene, fluoranthene, benzo falpyrene, benzo [g,h,i]fluoranthene, and dibenzo [cd,jklpyrene was prepared. All six compounds were purified by paper chromatography for use in this experiment. Emission spectra were obtained on this mixture a t excitation wave lengths from 250 to 400 mp a t lO-mp intervals. All the emission spectra from 250 to 300
U
x
a 3 1
N
T3
I
w
C
M W
m
ec,
' 9 3
W
d
x
VOL. 32, NO. 11, OCTOBER 1960
0
1439
Table V, Compound
Fluorescence Spectra
R - N A Emission
XiExcit.
A
QUINOLINE 330" -
317
317
ACRIDINE 40OC -
360
A
300
350
400
380 (6)
PHENANTHRIDINE
-
250
uv
-
335
345
350,367,385,400
DIBENZ [a,h]ACRIDISE
-
393
395,403,418,445,470 (395,405,417) - (IO)
300
-
450
W A V E L E N G T H , h4)
Figure 5. Fluorescence emission spectra of indole at 5.5 T/ml. I. 2. 3.
DIBEPZ[ U , ~ ] A C R I D I N E
In water In cyclohexane In ethyl alcohol
395,400,416,440,465 -
300
-
393
(392,403,415,426,439) ( I O )
mp and from 320 to 390 mp were identical and agreed with the fluorescence emission spectrum of benzo la jpyrene. The spectra obtained with excitation a t 310 and 400 mp agreed with the emisPion spectrum of dibenzo [cd,jre]pyrene. The other four compounds could not be detected in the presence of the strongly fluorescent benzo (a]pyrene. Since several useful methods are available for the paper chromatography of aromatic compounds, no further attempts were made to analyze complex mixtures directly by fluorescence spectroscopy. Solvent a n d pH Effects. -4number of compounds are known t o exhibit shifts in fluorescence emission maxima when the pH of the solutions is changed. Some of these compounds are used as fluorescent indicators for acid-base titrations. The isomeric compounds dibenz [ajlacridine and dibenz [a$]acridine show shifts to longer wave lengths in their emission spectra when dissolved in concentrated sulfuric acid as compared to their spectra in cyclohexane. Comparable shifts oceur in their fluorescence excitation and ultraviolet absorption spectra. The maxima in the spectra in cyclohexane and in sulfuric acid are shown in Table 11. These changes in spectra with p H were used as an aid in the identification of the two dibenzacridines in cigarette tars (16). I n some cases a change of fluorescence emission spectrum is not accompanied by any wave length shift in the ultraviolet absorption and fluorescence excitation spectra, Thus, the ultra1440
ANALYTICAL CHEMISTRY
c
10-mfi slits. Quinoline contained an impurity which showed fluorescence emission a t It could not be removed by column chromatography Values in parentheses are from the followed by distillation in a nitrogen atmosphere. literature ( I O ) . 6 20-mfi slits. a
400 mp (excitation at 317 mp).
Table VI.
Fluorescence Spectra of Tetracyclic and Higher Aromatic Hydrocarbons
Compound Yaphthacene Benz[a]anthracene Chrysene
A Excit.
A Emission
Xcv
AA
275 344 329
471(2) 385 ( 8 )
5 0
360(8) 340(2)
5
410
4i6,508, 550, (472, 506) ( I O ) _ 404,426,454(385,407,433) %, _ (10) 362 385,405, 430 (361,373, 381, 395, 4b3)(10) 34-60,- 400 (353) ( I O ) _ 391, 399, 378, - 400, 422,435, 452 (379, _ 4 1 1 32 2 ) (10) 330 355 342. 362 350: Z O (342,346,358,379) (10) 385, - 378, 383, - 387, - m2, 430, 445 (373, 392) (10) 427, - 428, 457, 485 (403, 408, 416, 405, 431237.453) ( l .o r 391 (391) ( I O ) ' 470, 550 430, z 5 , 460, 477, 485 449, 485, 5=(448,477) (10) 438,465,490 ( E O , 4 7 487) (10 ) KO_ ,466, 503 (437, 465, 497) ( I O ) -
385 341
405,426,450,476 435,447, 475,485,508,525 - %,-
406(6) -1 428(%) 7
318
370,387,405 (366,376,386,408) (IO) --
365
5
357
405,430,457,485 --
396
9
360
402,415,441,468
356
46
Triphenylene Picene
305 329
hcenaphthene 1,l'-Dinaphthyl 2,2'-Dinaphthyl 2-Phenylnaphthalene Pyrene
309 320 320 295 340
Benzo[a]pyrene
380
Benzo [ e ]pyrene Dibenzo [a,I]pyrene Dibenzo [a,i]pyrene Dibenzo [a,h]pyrene Dibenzo [cd,jk]pyrene Dibenz [de,kl]anthracene Benzo [g,h,z]perylene Dibenzo [ g h i , p p r ] perylene 11H-Naphtho[2,l-a]fluorene 13H-Naphtho [2,3-b]fluorene Fluoranthene
347 400 395 311 400
m,
'
376(2)
2
315(6) 333(6) 285
2 10 40 9 65
371 (8)
14
320
402 3 388 ( 2 ) 3 454 (2) 16 433 ( 2 ) -3 -2 45 1 433 ( 2 ) 5 434 ( 2 ) 6
of Naphthalene Series AX
X
R = CH Emission
Excit.
X
x
uv
AX
NAPHTHALENE 13
295
20
370
330,343,350 (322,325,333, _ _ 337,346,350I
ANTHRACENE
380,401,425,450 (3 x __ 399,422,448)
319
11
375
5
345
-1
395
2
PHEXIXTHRENE
5
344,360,379,400 ---
29 1
-
(347,357,364,376,383) -
DIBENZ [U,h]AXTHRACESE
2
300
397,406,420,440,454 (39q4OT41r444) -
-
DIBENZ [ a , j ]ASTHRACESE
2
...
(394)
.*.
...
~
Fluorescence Spectra of Tetracyclic and Higher Aromatic Hydrocarbons (continued) Compound Excit. A Emission AUV AX 350 425,452, 480, 507 419 6 Benz [g,h,i]_ Table VI.
fluoran thene Benzo [ k ]fluoranthene Xiethylbenz [ a ] anthracenes
402
404,429,460,490 -
400
1-
345
(389,411,435) (IO)
236789-
345 345 345 345 345 345 345 345 345
385,407,435,466 _ -
387,407,430,470 386,410, - 435,465, (387,408,435)(IO) 391, 413, 436,465 (391,413,437)(IO) 390,413, 435,460 (387,410,435) (IO) 395, KO,440,480 (388, 411) (IO) z 2 , 4 1 7 , 442,475 (388,411,437) (IO) 385, 407, _ _ 437,460 (389,411,437) (IO) 395,420,442,455,474(394,417) (IO) _.-
387.5 (7) 383 384 384 389 386 386 386 384 391
355
392, _ 416, _ 135,475 (405) ( I O )
390
2
379
393, _ 415, _ 442, 4i0
392
1
316
451, 480, 522 - _
452
312 342 360 368
450, - _4T9, 510 380, 410 440,465, 500 445, - _471
445 372 (6) 358 362
5 8 82 83
369
425, 442, 455, _ 475, _ 492
363
62
363
425, 450, 475 _-
360
65
101112-
7,12-Dimethylbenz[ a ]anthracene 20-hlethylcholanthrere 6-Methyldibenzo[cd,jklpyrene
6,lZ.Dimetliyldibenzo[cd,jklpyrene 2-hlethylpyrene
2-Methylfluoranthene 7-Methylfluoranthene 1.2.3-Trimethvl' fluoranthene 8,g-Dimethylfluoranthene
4 2.5 2 3 2 2 4 9 6 1 4
-1
violet absorption spectrum of a cyclohexane solution of indole shows ninxima at 259,263,277, and 287 mp. The same maxima appear in the spectrum of indole with water or with ethyl alcohol as solvent, but with these solvents the bands are not well resolved and appear as shoulders on the 263-mp band. The fluorescence emission spectrum of a cyclohexane solution of indole shows a maximum at 297 mp. I n ethyl alcohol, the emission maximum is shifted to 330 m p and when dissolved in distilled water, the maximum is shifted to 350 mp. Indole shows the same fluorescence excitation spectrum in all three solvents with a maximum at 282 mp: The fluorescence excitation and emission spectra of indole in these three solvents are shown in Figures 4 and 5, respectively. Udenfriend and coworkers (12) have shown that 5-hydroxytryptamine exhibits a shift in fluorescence emission in the presence of acid as compared to the fluorescence in a neutral solvent. I n this case also there is no corresponding shift in the ultraviolet absorption spectrum. Tryptamine does not show a corresponding shift in the fluorescence emission spectrum when dissolved in dilute acid (12). I n the present work indole in dilute acid showed the same fluorescence emission spectrum as in water. The indole derivative, carbazole, gave similar fluorescence emission spectra in both cyclohexane and in water. The importance of the solvent and p H in determining the appearance of a fluorescence spectrum is dramatically illustrated in the case of acridine. Acridine is known to fluoresce weakly in hydrocarbon solvents and more intensely in ethyl alcohol solution ( 8 ) , and this behavior has been ascribed t o hydrogen bond formation with a proton donating solvent (9). I n the present work, the fluorescence and ultraviolet absorption spectra of acridine were examined in cyclohexane, ethyl alcohol, water, 10% aqueous sulfuric acid, and in concentrated sulfuric acid. The ultraviolet absorption spectra in the first three solvents are very similar. I n dilute and in concentrated sulfuric acid, there is considerable enhancement in the L values. I n addition, the band at 248 nip is shifted t o 254 mp, A number of bands of longer nave length appear in the fluorescence excitation spectra of acridine in sulfuric acid. The ultraviolet absorption, fluorescence excitation, and emission maxima are shown in Table 111. The fluorescence efficiency increases in the order: cyclohexane N H and -0There is a bathochromic shift in the ultraviolet absorption spectrum when Diethylene is replaced by >XH. A similar shift is found in the fluorescence. Thus, carbazole shows fluorescence at a longer wave length than does fluorene. The corresponding oxygen
1442
ANALYTICAL CHEMISTRY
analog, dibenzofuran, which in its ultraviolet absorption spectrum is very similar to fluorene (both show two series of peaks below 300 mp), is also similar to fluorene in its fluorescence emission spectrum. I n the tetracyclic series, 5H-benzo [b]carbazole, 11H-benzo [blfluorene, and benzo [blnaphtho [2,3-d]furan, the same correspondence between ultraviolet absorption spectra and fluorescence emission spectra is observed. I n Table V are listed the fluorescence spectra of the naphthalene series and I
the effect of replacement of =AH by =S--. I n the pentacyclic systems the fluorescence spectra are very similar in the carbocyclic and heterocyclic series. I n Table VI, the fluorescence spectra of the tetracyclic and higher aromatic hydrocarbons are tabulated. Schoental and Scott (10) compared the fluorescence spectra of benz [a]anthracene and its monomethyl derivatives and found small shifts in the fluorescence emission maxima to longer wave lengths. particularly when the methyl group is in the 5 , 7 , or 12 position. Because of the relatively small shifts involved, it seemed desirable to repeat the study of the fluorescence spectra of this series. The results, together with the reported Values are shown i n Table VI. Most of the band maxima recorded agree well with the maxima reported by Schoental (10). More pronounced shifts were observed in the methyl derivatives of dibenzo[cd,jk]pyrene and fluoranthene (Table VI).
LITERATURE CITED
(1) Bowen, E. J:, Wokes, F “Fluorescence o,f S?luti?!:,? p. 9, Longmans
Green, Lonaon, IY)J;J.
( 2 ) Clar, E., “Aromatische Kohlenwasserstoffe,” 2nd ed., Springer-Verlag, Ber-
lin. 1952.
(3) Cook, J. W., Hewitt, C. L., Hieger, I., J . Chem. SOC.1933, 395.
Forster, T., “Fluoreszenz organischer Verbindugen,” p. 94, Vandenhoeck and Rumecht. Gottinrren. 1951. (5) Fbrster,’ T., Kasper, K., 2. Elektrochem. 59,976 (1955). (6) Friedel, R. .A,, Orchin, M.. “Ultraviolet Spectra of Aromatic Compounds,’] Wiley, New York, 1951. ( 7 ) Jones, R. N., J . Am. Chem. SOC. 62, (4)
148(1940). ~~
\ - -
(8) Mataga: N., Kaifu, Y., Koizumi, M., Bull. Chem. SOC.(Japan) 29, 373 (1956). (9) Mataga, S . , Tsumo, S., Ibid., 30, 368 (1957). (10) S‘choentnl, R., Scott, E. J . Y., J . Chem. So?. 1949, 1683. (11) Sprince. H., Rowlev, G. R.. Science “ I
125,-25(1957). (12) Udenfriend, S., Bogdanski, D. F., Keissbach, H., l b i d . , 122, 972 (1955). (13) Van Duuren, B. L., J . Natl. Cancer Inst. 21, l(1958). (14) Ibtd., p. 623. (15) Van Duuren, B. L., Bilbao, J. A., Joseph, C. A., Ibzd., 25, 53 (1960). (16) West, W.,“Techniques of Organic Chemistry,” Vol. IX, “Chemical Applications of Spectroscopy,” p. 716, -4. Weisshergw, ed., Interscience, New Tork, 1956 .
RECEIVED for revien. January 27, 1960. Accepted June 3, 1960. The spectrofluorometer used in this work was purchased through a field investigation grant, 90. CS-9577, from the Public Health Service, National Institutes of Health of the U. S. Department of Health, Education, and Welfare, National Cancer Inetitutp.
ACKNOWLEDGMENT
This work was supported by a grant from the American Cancer Society, Inc., S e w York. The author is indebted to Norton Selson for his interest and encouragement in this a o r k and to C. A. Joseph and J. A . Bilbao for technical assistance. Small samples of some of the aromatic compounds used in this work were kindly supplied to 11s by E. D. Bergman, Sg. Ph. BuuHoi, E. Clar, R. E . Dean, 1,. Z’,Fieser, M. Kloetzel, M. Newman, 11. Orchin, and R H. Tucker.
Correction The Sulfur Dioxide Test for Selenious Acid I n this article by Earle R. Caley and Clayton L. Henderson [ANAL.CHEJI.32, 9 i 5 (19SO)], the correct abbreviation for the journal in references 6 and S is Ann. Physik Chem., as given in the authors’ original manuscript.
