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techniques of obtaining
luminescence spectra have been
used by physical chemists for many
years, they have found little use in
analytical chemistry until recently.
One reason for this slow rate of application has been the lack of commercially
available instrumentation. However,
instruments are now available and the
photoelectric recording of fluorescence,
phosphorescence, and excitation spectra
is within the scope of most well-equipped
analytical laboratories. Fluorometry
has been used by analytical chemists
for some time but only recently has
spectrofluorometry with choice of excitation wavelengths been used for analysis
and
characterization
of
organic
materials. Parker and Rees (15) have
used the analogy that fluorometry is
related to spectrofluorometry as filter
photometry is related to absorption
spectrophotometry.
Spectrofluorometry offers a t least two
distinct advantages over absorption
spectrophotometry, uiz., greater sensitivity and several spectra (excitation
and emission) as criteria for identification. However, not all substances
which absorb radiation exhibit fluorescence.
Spectrophosphorimetry, which is
now beginning to be applied in
analytical chemistry, offers even more
information than spectrofluorometry
for the characterization and identificaLTHOUGH
10
ANALYTICAL CHEMISTRY
70827
11
Fluorescence
Phosphorescence
1 .O pg./ml.
Isooctane
Curve 4
Curve 2
Concn.
Solvent
Excitation
Emission
0.58 mg./ml.
EPA
Curve 3
Curve 1
~~~
~~
Luminescence Characteristics of
Reference Compounds. Typical ex-
12
ANALYTICAL CHEMISTRY
Compound
Tetrahydrocarbazole
Solvent
EPA
1,2-Dimethylindole
EPA
Dimethylthianthrene
EPA
Quinaldine
EPA
Excitation
wavePhosphorescence
lengths. Wavelength. Corrected
mp
mp
rel. intensity
292
411
0.36
224
439
0.53
0.44
460sh.
0.28
500b.sh.
414
288
0.31
441
0.44
454sh.
0.34
464sh.
0.33
285
422?
...
482
0.14
46 1
300
0.086
471sh.
0.059
0.13
493
0.11
529
0,042
568
0.0080
332
590
Y
5,7-Dimethylbenzacridine
EPA
2,2'-Dinaphthyl ether
EPA
279
315
1-Asapyrene
EPA
368
370
485
515
-
542sh.
524
561
-
o-Dianisidine
EPA
305
2-Amino4methylpyridine
2-Methylacridine
EPA
EPA
302
292
Diphenyl sulfide
Diphenyl ether
EPA
EPA
Diphenylamine
EPA
285
234
264
305
597
498
524
439
437
451sh.
464
416
404
40 1
417
427
0.16
0.27
0.20
0.00053
0,00055
0.00041
0.053
0.078
1.02
0.0012
0,0013
0.0014
Predominant
vibrational
spacing,
cm.-l
...
1580
...
...
...
1450
...
...
...
...
...
1400
1400
1500
...
...
...
1210
iib
1090
...
...
...
...
...
1300
1.4
...
0.44
...
3.6
2.8
2.5
...
...
...
(Continued)
Compound
Dithieiio (2,3-b,5,4b)thiophene
B ~ ~.( lJ2-b,5,4-b)z o
dithiophene
Solvent
PH
PH
Excitation
wavePhosphorescence
lengths, Wavelength. Corrected
rel. intensity
mw
mp
272
287
3-Methylbenzo ( b )
thiophene
PH
280
1-Thiaindane
2-Thiaindane
[2-Benzo ( b ) thienyllphenylthiamethane
PH
PH
PH
293
280
300
1,2,3,PTetrahydrothianaphtheno-(2,3-b)thianaphthene
Acridane
PH
297
EPA
308
465
459
466
476
494
ash.
511sh.
527sh.
440sh.
450sh.
466
497
PH
295
318
Carbazole
a-Naphthoquinoline
7,8-Benzoquinoline
1,2-Benzocarbazole
2-Methylindole
EPA
EPA
EPA
EPA
EPA
1-Methyl-2-phenylindole
EPA
314-Benzoquinoline
EPA
Diphenylene oxide
EPA
285
325
315
332
-
282
317
329
317
338
285
315
315
330
-
284
440
435
460sh.
485
515sh.
474
414
__
425
437
479
492
501
518
531
54lsh.
559
574
410
426
439
456
465sh.
466
475
501
538
575sh.
468
477
504
514sh.
540
580sh.
483
497
519
553sh.
408
420sh.
434
446sh.
458
500
531
568sh.
455
460sh.
488
523
560sh.
414
435sh.
444
461
469
479
0.0031
Predominant
vibrational
spacing,
cm.-l
...
1.1
1.1
1.0
1.1
1.0
0.97
0.88
0.145
0.16
0.28
0.27
0.069
300
430
1530
0.0001
...
...
...
...
...
...
...
...
...
1.6
2.8
2.8
0.62
...
...
...
...
1.5
1.3
...
1.2
0.31
0.28
0.20
0.44
0.38
0.27
0.27
0.19
1.0
0.55
0.99
0.55
0.52
0.047
0.036
0.064
0,045
0.045
0.19
0,090
0.23
...
0.17
0.034
0.031
0.063
0.042
0.38
0.27
0.44
0.30
0.28
0.011
0.019
0.012
0.12
0.11
0.20
0.17
0.078
0.75
0.50
0.81
0.45
0.44
0.42
600
680
...
500
...
1550
480
...
...
...
...
...
1600
...
...
...
...
1500
...
...
...
1600
...
...
...
...
...
1500
...
...
1500
...
...
...
...
...
...
...
1400
...
...
...
...
1650
...
...
(Cmkked)
13
PredomExcitainant
tion
vibrawavePhosphorescence
tional
lengths, Wavelength, Corrected spacing,
Compound
Solvent
mfi
mr
rel. intensity cm. -1
2,3-Benzodiphenylene oxide
EPA
318
520
0.094
...
337
528sh.
0.058
...
542
0,063
...
563
0.099
1460
588sh.
0.060
...
3-Methylisoquinoline
EPA
282
495sh.
...
310
515
o.ooi2
...
Wsh.
...
...
Older data (different 1P28 phototube)
Dibenzothiophene 5-dioxide
EPA
285
445
0.30
...
315
478
0.45
...
510
0.39
...
EPA
312
425
Dibenzothiophene
1.5
...
439
1.3
...
456
1.5
...
EPA
270
370sh.
Not
detnd.
p-Tolyl sulfone
.
..
385
Not detnd.
...
EPA
285
364
0.99
Phenothioxin-10-dioxide
382
1.2
iioo
400
0.87
...
475
PH
290
0,019
Naphthalene
...
487
0.012
. .
510
0,027
1450
549
0,019
...
PH
280
...
354sh.
Triphenylmethane
...
373
0.27
...
386
0.28
...
405sh.
0.19
...
EPA
285
480
0.13
Thianthrene
...
410
EPA
283
0.070
Indole
...
437
0.097
1550
450sh.
0,052
...
414
EPA
285
0.27
Methyl p-tolyl sulfide
*..
285
420
0.34
Ethyl p-tolyl sulfide
...
Very weak phosphorescence
EPA
Di-p-tolyl disulfide
...
Very weak phosphorescence
EPA
Diphenyl disulfide
...
Very weak phosphorescence
EPA
Dibenzyl disulfide
...
303
EPA
8-Meth ylquinoline
474
0.0069
,..
486
0.0045
...
0.0097
1400
508
546
0.0069
...
EPA
308
472
0.0014
Isoquinoline
550
484
0.0016
350
493
0.0025
500
506
0.0031
519
550
0.0030
0,0039
529
350
543
0.0030
500
458
0.028
EPA
Quinoline
298
...
470
0.020
550
490
0.036
1450
502sh.
0.025
...
526
0.027
...
0.025
EPA
310
473
2,6-Dimethylquinoline
...
0.022
482sh.
...
508
0.044
1450
544
0,033
...
429
2,2 -Bipyridine
EPA
295
0.025
...
449
...
0.025
1600
460
0.036
0.031
480
...
0.063
PH
240
389
Acetophenone
..*
1650
416
0.095
278
1750
320
448
0.070
0.041
1600
483
14
ANALYTICAL CHEMISTRY
Compound
Aromatic hydrocarbons
Acenaphthene
Indene
1-Methylnaphthalene
2,7-Dimethvlnaphthalkne
Oxygen compounds
Diphenyl ether
Dibenzofuran(diphenylene oxide)
2,3-Benxodiphenylene
oxide
Nitrogen compounds
Diphenylamine
Quinoline
Quinaldine
2-Methylacridine
Tetrahydrocarbazole
1,2-Dimethylindole
5,7-Dimethylbenzacridine
3,CBenzoquinoline
7,8-Benzoquinoline
2-Methylpyridine
CMethylpyridine
2,4,6-Trimethylpyridine
2,2-Bipyridine
Acridane
Carbazole
Mean
lifetime,
Sol- secvent onds
PH
PH
PH
2.8
0.5
2.0
PH
1.8
EPA 0.45
EPA 6 . 1
EPA 1 . 5
EPA
PH
EPA
EPA
EPA
EPA
1.9
0.8
1.7
3.8
4.8
5.2
EPA
EPA
EPA
PH
PH
PH
PH
EPA
EPA
0.26
1.2
2.1
1.3
1.7
1.8
1.0
3.0
8.1
Sulfur compounds
Di-p-tolyl sulfone
EPA 1 . 4
Phenothioxin 10dioxide
EPA 0.75
Diphenyl sulfide
EPA 0.034
Dimethylthianthrene
EPA 0.072
EPA 0.063
Methyl p-tolyl sulfide
Thianthrene
EPA 0.040
[2-Benzo ( b ) thienyl]
phenylthiamethane
PH 0.055
1-Thiaindane
PH 0.045
3-Methylbenzo ( b )
thiophene
PH 0.22
2,3-3,2-Dithianaphthene
P H 0.034
Benzo ( 1,2-b15,4b)dithiophene
PH 0.26
1,2,3,PTetrahydrothianaphthenoP H 0.074
(2,3-b) thianaphthene PH 0.074
a Time required to reach 36.8y0 of
initial intensity.
Theory, Interpretation, and Discussion of Spectra. I n the fluorescence process, the molecule is raised
from a singlet ground state ( 8 ) to an
or vibrational
excited singlet state (S*)
level thereof, followed by vibrational
deactivation to the zero vibrational
level of S*, thence via fluorescent emission to S or vibrational level thereof.
Since the vibrational levels of S and S*
are similar, the emission spectrum is
usually an approximate mirror image of
the absorption spectrum (12). Thus,
where fine structure may be observed,
something of the vibrational frequencies
in the molecules may be determined
and used as a means of characterization
or identification.
Phosphorescence involves a metahaving a longer
stable triplet state (T*)
lifetime and a lower energy than the
singlet excited state. Electronic transitions between two states involving a
change in multiplicity, as in the T* +
S phosphorescent emission, are normally
forbidden. The probability of transitions between these states, however, is
governed by the degree of spin-orbital
interaction (magnetic interaction between the spin and orbital motions of an
electron). I n atoms of low atomic
number the spin-orbital coupling is
small, while for heavier atoms it is
larger, The constant in the expression
describing this magnetic interaction is
proportional to the fourth power of the
effective nuclear charge. McClure (14)
found that substituting heavy atoms in
organic molecules produced the same
effect as observed in atoms. Although
this substitution causes little change in
the wavelength and intensity of the
absorption spectra, increased probability of T*-+ S transitions decreases
the lifetime in the T* state. For the
halogenated naphthalenes substituted
in the a-position, McClure (14) found
the following lifetimes for phosphorescence: F = 1.5, C1 = 0.30, Br =
0.018, and I = 0.0025 second.
Nitrogen and oxygen analogs of
aromatic hydrocarbons should exhibit
phosphorescence lifetimes equivalent to
their hydrocarbon analogs (Tables IV
and V). Sulfur compounds show much
shorter lifetimes and higher intensities
than the hydrocarbon or oxygen analogs
because of increased spin-orbital interaction (Table IV).
The phosphorescence intensities of
many of the oxygen, nitrogen, and
sulfur compounds shown in Table I11
are high compared to their fluorescence intensities (Table 11). This is
contrasted to the behavior of the hydrocarbon analogs which show weak
phosphorescence. It is for this reason
that spectrophosphorimetry becomes
useful in the characterization of sulfur
and nitrogen compounds, particularly
when micro separation techniques are
used and sensitivity is needed.
Table V.
Excitation
max ,, Emission max., mp
Phos.
Compound
mr Fluor.
280
340
...
Aniline
...
290
360
Methylaniline
...
255
370
Dimethylaniline
...
280
350
Indole
...
290
370
Skatole
...
355
280
2-Methylindole
...
. . . 404
Benz(c)acridine
. . . 406
...
11H-Benzo(a)carbazole
...
11H-Benzo(b)carbazole
...
408
396
...
7H-Benzo(c)carbazole
. . . . . . 372' *
Aniline
...
. . . 408, 438
Carbazole
...
. . . 865
Chlorophyll-b
...
. . . 493
Dimethyl-a-naDhthvl- amine
2,4-Dimethylquinoline
...
. . . 458
. . . . . . 420
p-Dimethyltoluidine
...
. . . 395, 412,
Diphenylamine
423
...
. . . 680-870
Metal-etioporphyrin11
complexes
400-600
...
...
Indole
462
...
Quinoline
400-600
323
...
Isoquinoline
660-760
,..
hletal-mesoporphyrinIX . . .
complexes
470
...
...
8-Meth ylquinoline
a-Naphthylamine
...
. . . 498
. . . . . . 498
p-Naphthylamine
502
...
...
Di-@-naphthylamine
. . . . . . 494, 540,
a-Naphthonitrile
587
... . . . 484
p-Naphthonitrile
. . . 495
Phenyl-p-naphthylamine . . .
335
350
...
Phenanthridine
367
385
400
300
...
395
Dibenz(a,h)acridine
403
418
445
470
300
...
395
Dibenx(a,j)acridine
400
416
440
465
280
348
...
Indole
256
322
...
Isoindazole
290
250
...
310
Benzimidazole
275
-
Hal flife
of phos.,
seconds
...
...
...
...
...
...
...
*..
...
...
Long
Long
10-4
Long
Long
...
Long
10-4
...
Long
5 X'io-4
4-Axaindole
5-Azaindole
6Axaindole
7-Axaindole
Benztriazole
3,4-Diaxindole
3,5-Diazaindole
Purine
Aniline
N,N-Dimethylaniline
N,N-Diethylaniline
Diphenylamine
Triphenylamine
Long
Long
Long
Long
Long
Long
Long
...
...
...
...
...
2.5gb
3.80b
...
4.45b
417
404
...
*..
...
...
1.096
0.276
392
...
...
2.16b
391
350
320
...
...
*..
...
...
...
0.026
4,466
...
...
...
...
355
370
33OC
336O
336c
345c
...
...
...
...
0.286
0. 14b
...
35OC
299
270
282
266
294
293
270
255
285
280
281
...
...
408
417C
412c
398"
415"
422c
410~
427"
435c
0.3gb
4.2
2.4
2.0
1.9
0.70
(6)
(Continued)
VOL. 38, NO. 1, JANUARY 1966
15
To ble V.
Compound
9,10-Dihydroacridine
Indole
Carbazole
Indole
Carbazole
5H-Benzo(b)carbazole
7H-Dibenzo(a,g)carbazole
7H-Dibenzo(c,g)
carbazole
Excitation
max., Emission max., mru
mp Fluor. Phos.
, , .
347~ 412O
. ..
439c
297c 403c
. . . 43lC
455c
. . . 342< 406c
357c 41SC
. . . 435c
280
297
...
32 1
335
...
348
330
387
...
411
440
340
367
386
408
430
350
363
...
382
400
424
330
...
317
400
360
...
297
285
306
280
315
280
291
315
...
282
320
...
290
337
306
Half life
of phos.,
seconds
1.6
Ref.
0.36
(6)
0.55
(6)
Remarks
(6)
. ..
...
(21)
(21)
...
(21)
IN A
LATE.
...
(21)
* ..
(21)
Quino1ine
...
(21)
Acridine
Indole
2-Methylindole
3-Methylindole
1,2-Dimethylindole
2,3-Dimethylindole
...
(82)
2-( 3-Indolyl)-2,3dihydroindole
(indole dimer)
360
..
(22)
1-Methyl-2-phenylindole 310
..
(22)
332
290
Carbazole
348
318
330
Ap roximate limit of detection in pg./ml.
b RePative intensity in water at p~ of maximum emission.
c Values read from reproduced spectra (relative intensity us. frequency).
d Relative intensity in cyclohexane (uncorrected).
55
50
45
35
40
30
6.0d
8.2d
64. Od
5.4d
25
TIME (MINUTES)
16
ANALYTICAL CHEMISTRY
430-650" F.
4'
150' C.
30 pl.
STRAIGHT-RUN
MIDDLEDISTILX 430-650" F. distillation
1.0
0.8
0.6
Y
m
<
0.4
IIAF"WE
(Mp)
Figure 4. Phosphorescence and excitation spectra of gas chromatographic fraction No. 4 of a basic nitrogen extract of a 430650" F. petroleum fraction
Curve
Curve
Curve
Curve
0.2
1.
2.
3.
4.
Figure 3. Ultraviolet spectrum of gas chromatographic fraction No. 4 of a basic nitrogen extract of
a 430-650" F. petroleum fraction
\
\
'-v
400
300
:\,
2(
WAVELENGTH (H?)
fractions. These compounds were expected in only the last several fractions.
Because this fluorescence occurred in all
fractions at about the same intensity, it
was concluded that the acridines were
bleeding from the column as a result of
the sample having been run several
times before trapping was done. These
data emphasize the fact that high-boiling
17
OC PEAK 2
GC PEAK 7
----wc.
-EXC.
a10ql
OC PEAKS 9 & 10
A -EXC.
---EXC. 250
288
298W
WAVELENWCK @)
Figure 7. Phosphorescence spectra of trapped gas chrornatographic fractions of product from catalytic hydrogenation of
quinoline
TIME (MIN.)
Figure 6. Gas chromatogram of nitrogen compounds
from catalytic hydrogenationof quinoline
Trapped fractions are indicated by number
Table VI.
Lifetime for Phosphorescence of Trapped Fractions
1.2
1.0
1.1
1.4
1
2
3
4
0.9
0.8
1.1
1.3
1.3
1.4
5
6
7
8
9
1.2
1.2
1.5
1.2
1.4
13
14
15
1.2
12
Total sample
1.2
1.4
1.3
1.3
Reference compounds
Quinoline
No.emis2-Methylpyridine
sion
1. 3
PMethylpyridine
1.7
2,4,6-Trimethylpyridine
1.8
18
ANALYTICAL CHEMISTRY
n.6
0.3
13
0.6
1.0
15
0.7
Intensity of emission from pyridines
and tetrahydroquinolines.
b Intensity of emission from quinolines,
14
VIII.
catalytic
alkyl chain
transfer
catalytic
J.
Zmethylquinoline
2-ethylquinoline
Zisopropylquinoline
I
catalyst
0.8
No. emission
No.emission
No.emission
~
11
12
hydrogen
(by transfer)
1.2
1.3
1.2
1.1
1.2
uinoline
1.2
1.2
1.0
1.1
1.4
1.2
10
11
ACKNOWLEDGMENT
GC
peak
No.
1
2
3
4
5
6
7
8
9
10
11
12
(lG)Ibid., p. 30.
(20) Supplemental DBta Sheet to Aminco
Bulletin No. 2278, American Instrument Co., Inc., Silver Spring, Md.,
Atxi1 1959.
(21)Van Duuren, B. L., ANAL. CHEM.
32, 1436 (1960).
(22) Van Duuren, B. L., J . Org. Chem. 26,
2954 (1961).
(23) Yoshida, T., Igaku Kenkyu 27,
443 (1957); C . A . 52, 10721i (1958).
RECEIVED
for review February 18, 1965.
Accepted October 7, 1965. Presented at
the Symposium on Sulfur, Nitrogen, and
Oxygen
Compounds, Division of
Petroleum Chemistry, 149th Meeting,
ACS, Detroit, April 1965.
L. SOMMERS
Esso Research laboratories, Humble Oil & Refining Co., Baton Rouge, l a .
70821
19