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The spectrofluorqmetric determination of anthracene, fluorene and phenanthrene in mixtures
Talanta. 1961. Vol. 7. pp. 181 to 186. Pergamon Press Ltd. Printed in Norrhern Ireland
THE SPECTROFLUOROMETRIC DETERMINATION OF ANTHRACENE, FLUORENE AND PHENANTHRENE IN MIXTURES GLENN A. TIIOMMJB* and ELMER LEININGW Kedzie Chemical Laboratory, Michigan State University, East Lansing, Michigan, U.S.A. (Received 8 Jury 1960) Summa~-A procedure is presented for the spectrofluorometric determination of mixtures of anthracene, fluorene and phenanthrene. The determination depends on differences in fluorescence emission spectra and on selective excitation of anthracene fluorescence. Some of the fluorescence and absorption spectra involved overlap, but these difficulties can be overcome by empirical corrections. The average relative error in this method is less than 5 % over the concentration range 0 to 5 ppm.
use of the spectrofluorometer has greatly facilitated the fluorometric resolution of mixtures of compounds, particularly those depending on selective excitation and significant differences in fluorescent emission spectra. The fluorescence spectra, uncorrected for instrumental differences of sensitivity with wavelength, of anthracene, fluorene and phenanthrene in absolute methyl THE
I 25 -
I
I
1
I
I
I
I
A- FLUORE NE SPECTRUM 265 rT-&l EXCITATION
M
B-PHENANTHRENE SPECTRUM 265 ftly EXCITATION C- ANTHRACENE SPECTRUM 365m)J EXCITATION C
I5
-
IO -
5-
Q 290
I 310
I 350 WAVELENGTH
FIG. I.-Fluorescence
390
430 EMISSION
470
(rnp)
spectra of hydrocarbons
alcohol solution are shown in Fig. 1. Casual observation suggests a possible resolution of a mixture of these hydrocarbons in the following manner: (a) Only anthracene is fluorescent under 365 mYexcitation. Therefore, irradiation at this wavelength and measurement of the fluorescence intensity at 400 m,u should yield, directly, the anthracene concentration. * Present address: Jersey, U.S.A.
E. I. du Pont de Nemours
and Co., Inc. Photo Products 181
Department,
Parlin,
New
182
GLENNA. THOMMES and ELMERLEMNGER
(b) Although excitation with 265 mpradiation causes all three compounds to fluoresce, the fluorescence intensity measured at 316 rnp should be a measure of the fluorene concentration. (c) At 350 rnp, under 265 m+rradiation, a fluorescence intensity measurement should be the sum of the phenanthrene and fluorene emissions at this wavelength. Since the fluorene concentration would be known from (b), the phenanthrene concentration could be determined providing the fluorescence intensities are additive. In practice, however, the resolution is complicated by the fact that the absorption spectra of anthracene and phenanthrene3 interfere. Anthracene absorbs at 350 rnp and both anthracene and phenanthrene absorb at 316 rnp. Consequently, only the anthracene emission at 404 m,u, under 365 rnp-excitation, is free from interference and the fluorescence readings at both 3 16 and 350 rnp are dependent on the concentrations of all three compounds. Since the concentration of anthracene can be measured independently, a correction can be made for the interaction of anthracene with the fluorescent emission of the other hydrocarbons at 350 and 316 rnp. For example, if the assumption is made that the interaction is only due to absorption, the observed fluorescence intensity, Ip, is equal to the fluorescence intensity in the absence of anthracene, If’, minus the intensity absorbed by the anthracene present. Thus: I,a = I,” - I,“(1 - ~O-KACA) = If@x lO-%c~ where KA is the product of the molar absorptivity of anthracene at the wavelength of fluorescence measurement and the path length of the fluorescent emission, and C, is the molar concentration of anthracene. If the above assumptions were correct, a plot of log I,” - log Ipa versus C, should be linear. Two such plots, showing the effect of anthracene on the fluorescence of fluorene at 350 and 316 rnp are shown in Figs. 2 and 3. Although the plots are not linear, they may be used for correction purposes. The curves were obtained by observing the decrease in the fluorescence intensity of fluorene at 316 and 350 my as a function of anthracene concentration. The decrease was only a function of the anthracene concentration and was inde~ndent of the fluorene concentration. Furthermore, the 350 rn~-co~ection is also satisfactory for use with phenanthrene fluorescence. With the above information and the appropriate calibration curves, mixtures of anthracene and phenanthrene or anthracene and fluorene can be resolved easily. Mixtures containing phenanthrene and fluorene appear to be more difficult because of the mutual dependence of their fluorescence intensities at both 316 and 350 rnp. However, a simple graphical method4 can be used to resolve this difficulty. Any combination of 316 rnp- and 350 mp-fluorescence intensities must be unique for the concentrations of phenanthrene and fluorene involved. The fluorescence intensities of a series of fluorene and phenanthrene mixtures were measured at 316 and 350 m,u. These were plotted against each other and the points corresponding to identical fluorene concentrations were joined. Likewise, the points corresponding to identical phenanthrene concentrations were joined. The resulting calibration grid,
Determination of anthracene. tluorene and phenanthrene in mixtures
ANTHRACENE CONCENTRATION U’PM.) FIG. 2.-Anthracene
I ”
”
absorption of 350 mp emission
”
’ “““‘I
006-
ANTHRACENE CONCENTRATION IPP M.) FIG. 3.-Anthracene
absorption of 316 mp emission
183
184
GLENN A. THOMME~ and ELMERLEININGER
shown in Fig. 4, serves as a standard calibration curve for both fluorene and phenanthrene. If anthracene is present the 316- and 350 mp-measurements must be corrected for anthracene absorption before using the calibration grid. 90
80 5 E 70 ul 5 - 60 = 950
W
!s W
40
isi
30 Z 82 20 LL IO g
0
0
2
4
6
8
IO
12
14
16
FLUORESCENCE INTENSITY (350 mJJ) FIG. 4.-Calibration
grid
EXPERIMENTAL Reagents The anthracene and phenanthrene were purified by azeotropic distillation, under reduced pressure, with ethylene glycol and diethylene glycol according to the procedure of Feldman er aL2 The fluorene was purified by repeated vacuum sublimation. The starting materials in all of the purification procedures were Eastman White Label Chemicals. Baker’s analysed absolute methanol was used as the hydrocarbon solvent. Stock solutions of the three purified hydrocarbons were prepared which contained 05 mg of hydrocarbon per ml of solution. All subsequent solutions used were prepared by direct methanolic dilution of these stock solutions. Apparatus The spectrofluorometer employed was that described by the writers in a previous study.5 The instrument was calibrated each time before use with a standard solution containing 3.50 ppm of anthracene. The instrument was set to read 17.0 at 400 m,u under 365 my-excitation. The methanolic solution of anthracene was chosen as a calibration standard because of the well known oxygen quenching of the fluorescence of polynuclear hydrocarbons. Is8 Employing a standard which is also subject to oxygen quenching should eliminate any effect of day-to-day fluctuations in oxygen concentration. Calibration graphs A series of solutions of the three hydrocarbons was prepared to cover the range from 0.5 to 5.0 ppm. Each solution was made by transferring an aliquot portion of the appropriate standard stock solution to a 100 ml-volumetric flask, diluting to the mark with absolute methyl alcohol and mixing thoroughly.
~te~nation
of anthracene,
fluorene and ph~anthtene
in mixtures
185
The anthracene readings were obtained by measuring the fluorescence intensities of the anthracene solutions at 400 rnp under 365 my-excitation. The anthracene calibration curve was prepared from these data. The calibration grid for phenanthrene and fluorene was prepared by measuring fluorescence intensities at 350 rnp and 316 mp under 265 rnp-excitation of mixtures of phenanthrene and fluorene. The mixtures were prepared to cover the ranges of O-5 ppm of phenanthrene and O-5 ppm of fluorene. The correction curve for anthracene absorption was prepared as stated before. Calibration curves of the three hydrocarbons show linearity over the concentration range of O-5 ppm; consequently, this was used as the upper concentration limit in this study. However, the plots are smooth curyes above 5 ppm and the concentration range could probably be extended. Procedure
A series of unknown solutions were prepared comprising the four types of possible mixtures. These were prepared by dilution of aliquot portions drawn from standard stock solutions. The solutions were then excited with 365 rnp-radiation and the fluorescence intensity, if any, was measured at 400 rnp. This measurement is used dire&y with the anthraeene calibration curve, to obtain the concentration of anthracene present. The wavelength of excitation is changed to 265 rnp and fluorescence intensity measurements are made at 316 and 350 rnp. These measurements are corrected for anthracene absorption, if it is present, and are used with the calibration grid to obtain the concentrations of phenanthrene and fluorene present. If initial measurements indicate that the concentration of any of the hydrocarbons is greater than 5 ppm, a suitable dilution with methyl alcohol can be made to bring the concentrations into the proper range. TABLE I. RESULTS OF HYDROCARBON
T---
Anthraeene
MIXTURE ANALYSIS
Phenanthrene
Fluorene
---
I-
Taken,
Taken,
Found,
PPm
PPm
PPm
Percent error
4.00 l*oO 1.00 2+IO 3,OO 350 350 5.00 200 3.00 200 1.00 3.00 4.00
4.06 1.05 0.85 1.90 2.92 3.75 3.50 5.05 1.78 2-93 2.10 0.99 3.00 4.34
$-I.5 $5.0 -15.0 -5.0 -2.7 +7*1 0 -1.0 -11.0 -2.3 + 5.0 -1.0 0 +8.5
Taken,
Found,
PPm
PPm
Percent error
-
-
_075 :::
I : 1
2.00 3.00
0.73 144 1.00
’
1.95 2.95
-
j
--
I I
-
-
-2.6 -4.2 0 -2.5 -1.7
I
-
i
-
I
-
-
i
-
3.00 3.00 l+IO 3.00 2.00 1GO l*OO 250 3.80 3.00 2.00 200 1QO 3.00 l*Oo
3.07 2.14 1.05 3.04 20.5 0.98 1.00 252 3.45 3.15 220 2.20 1.02 3.20 1.05
$2.3 +7.0 +5-o +1.3 t2.5 -2.0 0 to.8 -1.4 +5.0 +10,0 + 10.0 +2.0 +6.7 -t-5.0
L
RESULTS
The results obtained on the mixtures prepared are summarised in Table I. The average errors found in the determinations of the three hydrocarbons were as follows : Anthracene, E._ = l-9 % Phenanthrene, E_ = 4.6% Fluorene, E,_ = 4-l %
186
GLENN
A.
THOMMESand ELMER LEININGER
The maximum error observed was 15 % ; however, errors of this order of magnitude are exceptional and in the majority of determinations the error is less than f 5 %. Acknowledgement-The authors are indebted to the Eastman Kodak Company for financial support during this investigation. Zusammenfassung-Ein Methode zur spektrofluorimetrischen Bestimmung von Anthracen, Phananthren und Fluoren wird mitgeteilt. Die Bestimmung beruht auf den Unterschieden in den Fluoreszenspektren und der selektiven Anregung der Anthracentluorescenz. Einige der Fluoreszena und Absortionsspektren iiberschneiden sich, jedoch kann diese Schwierigkeit durch Anbringen einer empirischen Korrektur tiberwunden werden. Der mittlere relative Fehler der Methode ist weniger als 5 % im Konzentrationsbereich O-5 ppm. RCsurn&Les auteurs presentent un pro&d6 de dosage spectrofluorom&ique de melanges d’anthrac&e, de phenanthrene et de fluorene. Le dosage depend des differences des spectres d’Cmission fluorescents et de l’excitation selective de la fluorescence de l’anthracene. Certains des spectres de fluorescence et d’absoprtion consider&s se recouvrent, mais ces difficult& peuvent Btre surmontees par des corrections empiriques. L’erreur relative moyenne dans cette methode est inferieure a 5 pour cent darts le domaine de concentration O-5 p.p.m. REFERENCES 1 E. J. Bowen and A. H. Williams, Trans. Faraaizy Sot., 1939, 35,165. 2 J. Feldman, P. Panteges and M. Orchin, J. Amer. Chem. Sot., 1951, 73, 4341. 3 R. A. Friedel and M. Orchin, Ultraviolet Spectra of Aromatic Compounds. John Wiley and Sons, Inc., New York, 1950 * J. W. Collat and L. B. Rodgers, Analyt. Chem., 1955, 27, 961. 5 G. A. Thommes and E. Leininger, ibid., 1958, 30, 1361. 6 H. Weil-Malherbe and J. Weiss, Nature, 1942, 149, 172; 1943, 151, 449; J. Chem. Sot., 1944, 541 and 544.
THE SPECTROFLUOROMETRIC DETERMINATION OF ANTHRACENE, FLUORENE AND PHENANTHRENE IN MIXTURES GLENN A. TIIOMMJB* and ELMER LEININGW Kedzie Chemical Laboratory, Michigan State University, East Lansing, Michigan, U.S.A. (Received 8 Jury 1960) Summa~-A procedure is presented for the spectrofluorometric determination of mixtures of anthracene, fluorene and phenanthrene. The determination depends on differences in fluorescence emission spectra and on selective excitation of anthracene fluorescence. Some of the fluorescence and absorption spectra involved overlap, but these difficulties can be overcome by empirical corrections. The average relative error in this method is less than 5 % over the concentration range 0 to 5 ppm.
use of the spectrofluorometer has greatly facilitated the fluorometric resolution of mixtures of compounds, particularly those depending on selective excitation and significant differences in fluorescent emission spectra. The fluorescence spectra, uncorrected for instrumental differences of sensitivity with wavelength, of anthracene, fluorene and phenanthrene in absolute methyl THE
I 25 -
I
I
1
I
I
I
I
A- FLUORE NE SPECTRUM 265 rT-&l EXCITATION
M
B-PHENANTHRENE SPECTRUM 265 ftly EXCITATION C- ANTHRACENE SPECTRUM 365m)J EXCITATION C
I5
-
IO -
5-
Q 290
I 310
I 350 WAVELENGTH
FIG. I.-Fluorescence
390
430 EMISSION
470
(rnp)
spectra of hydrocarbons
alcohol solution are shown in Fig. 1. Casual observation suggests a possible resolution of a mixture of these hydrocarbons in the following manner: (a) Only anthracene is fluorescent under 365 mYexcitation. Therefore, irradiation at this wavelength and measurement of the fluorescence intensity at 400 m,u should yield, directly, the anthracene concentration. * Present address: Jersey, U.S.A.
E. I. du Pont de Nemours
and Co., Inc. Photo Products 181
Department,
Parlin,
New
182
GLENNA. THOMMES and ELMERLEMNGER
(b) Although excitation with 265 mpradiation causes all three compounds to fluoresce, the fluorescence intensity measured at 316 rnp should be a measure of the fluorene concentration. (c) At 350 rnp, under 265 m+rradiation, a fluorescence intensity measurement should be the sum of the phenanthrene and fluorene emissions at this wavelength. Since the fluorene concentration would be known from (b), the phenanthrene concentration could be determined providing the fluorescence intensities are additive. In practice, however, the resolution is complicated by the fact that the absorption spectra of anthracene and phenanthrene3 interfere. Anthracene absorbs at 350 rnp and both anthracene and phenanthrene absorb at 316 rnp. Consequently, only the anthracene emission at 404 m,u, under 365 rnp-excitation, is free from interference and the fluorescence readings at both 3 16 and 350 rnp are dependent on the concentrations of all three compounds. Since the concentration of anthracene can be measured independently, a correction can be made for the interaction of anthracene with the fluorescent emission of the other hydrocarbons at 350 and 316 rnp. For example, if the assumption is made that the interaction is only due to absorption, the observed fluorescence intensity, Ip, is equal to the fluorescence intensity in the absence of anthracene, If’, minus the intensity absorbed by the anthracene present. Thus: I,a = I,” - I,“(1 - ~O-KACA) = If@x lO-%c~ where KA is the product of the molar absorptivity of anthracene at the wavelength of fluorescence measurement and the path length of the fluorescent emission, and C, is the molar concentration of anthracene. If the above assumptions were correct, a plot of log I,” - log Ipa versus C, should be linear. Two such plots, showing the effect of anthracene on the fluorescence of fluorene at 350 and 316 rnp are shown in Figs. 2 and 3. Although the plots are not linear, they may be used for correction purposes. The curves were obtained by observing the decrease in the fluorescence intensity of fluorene at 316 and 350 my as a function of anthracene concentration. The decrease was only a function of the anthracene concentration and was inde~ndent of the fluorene concentration. Furthermore, the 350 rn~-co~ection is also satisfactory for use with phenanthrene fluorescence. With the above information and the appropriate calibration curves, mixtures of anthracene and phenanthrene or anthracene and fluorene can be resolved easily. Mixtures containing phenanthrene and fluorene appear to be more difficult because of the mutual dependence of their fluorescence intensities at both 316 and 350 rnp. However, a simple graphical method4 can be used to resolve this difficulty. Any combination of 316 rnp- and 350 mp-fluorescence intensities must be unique for the concentrations of phenanthrene and fluorene involved. The fluorescence intensities of a series of fluorene and phenanthrene mixtures were measured at 316 and 350 m,u. These were plotted against each other and the points corresponding to identical fluorene concentrations were joined. Likewise, the points corresponding to identical phenanthrene concentrations were joined. The resulting calibration grid,
Determination of anthracene. tluorene and phenanthrene in mixtures
ANTHRACENE CONCENTRATION U’PM.) FIG. 2.-Anthracene
I ”
”
absorption of 350 mp emission
”
’ “““‘I
006-
ANTHRACENE CONCENTRATION IPP M.) FIG. 3.-Anthracene
absorption of 316 mp emission
183
184
GLENN A. THOMME~ and ELMERLEININGER
shown in Fig. 4, serves as a standard calibration curve for both fluorene and phenanthrene. If anthracene is present the 316- and 350 mp-measurements must be corrected for anthracene absorption before using the calibration grid. 90
80 5 E 70 ul 5 - 60 = 950
W
!s W
40
isi
30 Z 82 20 LL IO g
0
0
2
4
6
8
IO
12
14
16
FLUORESCENCE INTENSITY (350 mJJ) FIG. 4.-Calibration
grid
EXPERIMENTAL Reagents The anthracene and phenanthrene were purified by azeotropic distillation, under reduced pressure, with ethylene glycol and diethylene glycol according to the procedure of Feldman er aL2 The fluorene was purified by repeated vacuum sublimation. The starting materials in all of the purification procedures were Eastman White Label Chemicals. Baker’s analysed absolute methanol was used as the hydrocarbon solvent. Stock solutions of the three purified hydrocarbons were prepared which contained 05 mg of hydrocarbon per ml of solution. All subsequent solutions used were prepared by direct methanolic dilution of these stock solutions. Apparatus The spectrofluorometer employed was that described by the writers in a previous study.5 The instrument was calibrated each time before use with a standard solution containing 3.50 ppm of anthracene. The instrument was set to read 17.0 at 400 m,u under 365 my-excitation. The methanolic solution of anthracene was chosen as a calibration standard because of the well known oxygen quenching of the fluorescence of polynuclear hydrocarbons. Is8 Employing a standard which is also subject to oxygen quenching should eliminate any effect of day-to-day fluctuations in oxygen concentration. Calibration graphs A series of solutions of the three hydrocarbons was prepared to cover the range from 0.5 to 5.0 ppm. Each solution was made by transferring an aliquot portion of the appropriate standard stock solution to a 100 ml-volumetric flask, diluting to the mark with absolute methyl alcohol and mixing thoroughly.
~te~nation
of anthracene,
fluorene and ph~anthtene
in mixtures
185
The anthracene readings were obtained by measuring the fluorescence intensities of the anthracene solutions at 400 rnp under 365 my-excitation. The anthracene calibration curve was prepared from these data. The calibration grid for phenanthrene and fluorene was prepared by measuring fluorescence intensities at 350 rnp and 316 mp under 265 rnp-excitation of mixtures of phenanthrene and fluorene. The mixtures were prepared to cover the ranges of O-5 ppm of phenanthrene and O-5 ppm of fluorene. The correction curve for anthracene absorption was prepared as stated before. Calibration curves of the three hydrocarbons show linearity over the concentration range of O-5 ppm; consequently, this was used as the upper concentration limit in this study. However, the plots are smooth curyes above 5 ppm and the concentration range could probably be extended. Procedure
A series of unknown solutions were prepared comprising the four types of possible mixtures. These were prepared by dilution of aliquot portions drawn from standard stock solutions. The solutions were then excited with 365 rnp-radiation and the fluorescence intensity, if any, was measured at 400 rnp. This measurement is used dire&y with the anthraeene calibration curve, to obtain the concentration of anthracene present. The wavelength of excitation is changed to 265 rnp and fluorescence intensity measurements are made at 316 and 350 rnp. These measurements are corrected for anthracene absorption, if it is present, and are used with the calibration grid to obtain the concentrations of phenanthrene and fluorene present. If initial measurements indicate that the concentration of any of the hydrocarbons is greater than 5 ppm, a suitable dilution with methyl alcohol can be made to bring the concentrations into the proper range. TABLE I. RESULTS OF HYDROCARBON
T---
Anthraeene
MIXTURE ANALYSIS
Phenanthrene
Fluorene
---
I-
Taken,
Taken,
Found,
PPm
PPm
PPm
Percent error
4.00 l*oO 1.00 2+IO 3,OO 350 350 5.00 200 3.00 200 1.00 3.00 4.00
4.06 1.05 0.85 1.90 2.92 3.75 3.50 5.05 1.78 2-93 2.10 0.99 3.00 4.34
$-I.5 $5.0 -15.0 -5.0 -2.7 +7*1 0 -1.0 -11.0 -2.3 + 5.0 -1.0 0 +8.5
Taken,
Found,
PPm
PPm
Percent error
-
-
_075 :::
I : 1
2.00 3.00
0.73 144 1.00
’
1.95 2.95
-
j
--
I I
-
-
-2.6 -4.2 0 -2.5 -1.7
I
-
i
-
I
-
-
i
-
3.00 3.00 l+IO 3.00 2.00 1GO l*OO 250 3.80 3.00 2.00 200 1QO 3.00 l*Oo
3.07 2.14 1.05 3.04 20.5 0.98 1.00 252 3.45 3.15 220 2.20 1.02 3.20 1.05
$2.3 +7.0 +5-o +1.3 t2.5 -2.0 0 to.8 -1.4 +5.0 +10,0 + 10.0 +2.0 +6.7 -t-5.0
L
RESULTS
The results obtained on the mixtures prepared are summarised in Table I. The average errors found in the determinations of the three hydrocarbons were as follows : Anthracene, E._ = l-9 % Phenanthrene, E_ = 4.6% Fluorene, E,_ = 4-l %
186
GLENN
A.
THOMMESand ELMER LEININGER
The maximum error observed was 15 % ; however, errors of this order of magnitude are exceptional and in the majority of determinations the error is less than f 5 %. Acknowledgement-The authors are indebted to the Eastman Kodak Company for financial support during this investigation. Zusammenfassung-Ein Methode zur spektrofluorimetrischen Bestimmung von Anthracen, Phananthren und Fluoren wird mitgeteilt. Die Bestimmung beruht auf den Unterschieden in den Fluoreszenspektren und der selektiven Anregung der Anthracentluorescenz. Einige der Fluoreszena und Absortionsspektren iiberschneiden sich, jedoch kann diese Schwierigkeit durch Anbringen einer empirischen Korrektur tiberwunden werden. Der mittlere relative Fehler der Methode ist weniger als 5 % im Konzentrationsbereich O-5 ppm. RCsurn&Les auteurs presentent un pro&d6 de dosage spectrofluorom&ique de melanges d’anthrac&e, de phenanthrene et de fluorene. Le dosage depend des differences des spectres d’Cmission fluorescents et de l’excitation selective de la fluorescence de l’anthracene. Certains des spectres de fluorescence et d’absoprtion consider&s se recouvrent, mais ces difficult& peuvent Btre surmontees par des corrections empiriques. L’erreur relative moyenne dans cette methode est inferieure a 5 pour cent darts le domaine de concentration O-5 p.p.m. REFERENCES 1 E. J. Bowen and A. H. Williams, Trans. Faraaizy Sot., 1939, 35,165. 2 J. Feldman, P. Panteges and M. Orchin, J. Amer. Chem. Sot., 1951, 73, 4341. 3 R. A. Friedel and M. Orchin, Ultraviolet Spectra of Aromatic Compounds. John Wiley and Sons, Inc., New York, 1950 * J. W. Collat and L. B. Rodgers, Analyt. Chem., 1955, 27, 961. 5 G. A. Thommes and E. Leininger, ibid., 1958, 30, 1361. 6 H. Weil-Malherbe and J. Weiss, Nature, 1942, 149, 172; 1943, 151, 449; J. Chem. Sot., 1944, 541 and 544.