On
uae
George H. Schenk and Department of Chemistry, Wayne State Uniuersity9 Detroit, Mich.48202 If the lowest energy electronic transition of anthracene c ~ u l dbe removed, other aromatic hydrocarbons could be selectively excited in the presence of anthracene. The Diels-Alder reaction of maleic anhydride with anthracene can be used in practice to accomplish this. The Diels-Alder adduct of maleic anhydride and is not f ~ ~ o r e ~ c eand n ~ this , permits the c m e a s u r ~ ~of~ pyrene, n ~ diph perylene in the presence of the ilter effect of the a uct was negligible at c o n ~ ~ n t r a t i oat ~ s or below W . These findings extend the usefulness of the filter fluorometer for trace analysis of mixtures containing anthracene.
ANTHRACENE is a troublesome molecule in the fluorometric analysis of aromatic hydrocarbon mixtures because of the widely varying energies of its excited states. It has a broad excitation spectrum that ranges from 200 to about 370 mm, and a fairly broad fluorescence emission spectrum ranging from 365 to 490 mm. Thus, it will almost always be excited in any mixture, and will emit over a wide enough range to overlap the fluorescence bands of most aromatic hydrocarbons. In particular, the lowest energy excitation band (290 to 370 nm) of anthracene occrirs in a region where a number of aromatic hydrocarbons might be selectively excited if anthracene were not present. A rationale for the very low energy of the above band o f anthracene can be partially given in purely electronic terms, using a one-electron energy diagram (Figure 1) with Huckel energies ( I ) . The theoretical energy of 0.828 /? (see caption of Figure 1) for the lowest energy electronic transition of anthracene is quite low (less than half) compared to the theoretical energy of 2 p for the comparable transition of benzene. In addition, the lowest energy absorption (excitation) band of benzene should therefore occur at short wavelengths; it would not be expected to overlap the 290-370 nm band of anthracene. In fact, benzene does have an intense absorption band at 200 nm; it also has a band at 254 nm but this band is very weak (e = IOz>. For the purpose of fluorometric analysis, it is obvious that if the middle ring of anthracene can be reduced to a saturated ring, the molecule should behave like two benzene rings joined by methine groups. Theoretically then, fluorescence excitation should occur at higher energies than the 290-370 nm band of anthracene, facilitating fluorescence excitation of other aromatic hydrocarbons in the 290-370 nm region without interference. The ideal reaction for this purpose is the Diels-Alder reaction shown below (Equation 1).
(1) A. Streitweiser, “Molecular Orbital Theory for Organic Chemists,” Wiley, New York, N. Y . , 1961. 1754
e
This reaction involves the addition of a dienophile across the diene moiety of the middle ring of anthracene. An attempt has been made in our laboratories ( 2 ) to use tetracyanoethylene (TCNE) for this purpose. However, excitation even at 365 nm photodecomposed the Diels-Alder adduct back to anthracene and TCNE. In the present study, we chose maleic anhydride as a dienophile in hopes that its anthracene adduct would prove more stable to ultraviolet radiation and that its lower reactivity (as compared to TCNE) could be somehow increased. Another potential analytical difficulty is the possibility of fluorescence of the adduct if excitation at the 254-nm mercury line is to be used. It is clear from Figure 1 that excitation in this region should excite the adduct even as benzene itself is excited. Fortunately, benzene fluoresces very weakly when excited at 254 nm, and has a quantum efficiency of 0.042 (31,leading one to predict that the adduct would also fluoresce weakly and perhaps phosphoresce as benzene does. A final difficulty is the inner filter effect at 254 nm (4). Though it may not emit strongly, the adduct can still absorb enough ultraviolet radiation to cause a deviation from linearity in the fluorescence calibration curve for other aromatic hydrocarbons. The object of this study was to investigate all of the above points in connection with fluorometric measurement of hydrocarbon mixtures. EXPERIMENTAL
Apparatus. The Turner Model 110 filter fluorometer was used to follow the reaction of anthracene with maleic anhydride as well as to prepare calibration curves for the determination of other aromatic hydrocarbons after the reaction of anthracene. The 4-Watt general purpose mercury lamp was used for excitation at 365 nm, and the 4Watt far ultraviolet mercury lamp was used for excitation at 254 nm. The filter systems used for the various measurements were: first, for anthracene alone, anthracene-pyrene, and anthracene-diphenylstilbene mixtures, the 7-60 (peaks at 360 nm) narrow pass primary filter and as secondary filter, either a 405 (peaks at 405 nm) narrow pass filter or a Bausch and Lomb interference filter peaking at 394 nm with a half width of 15 nm [When the far ultraviolet source was used, the 7-54 (peaks at 254 nm) narrow pass primary filter was used with the above interference filter.], and second for anthracene-perylene mixtures, the 7-60 narrow pass primary filter, and as secondary filter, the 2-A sharp cut (415 nm) filter. When the far ultraviolet source was used, the 7-54 narrow pass primary filter was used with the 2-A sharp cut filter. The phosphorescence excitation and emission spectra of the Diels-Aider adduct of anthracene and maleic anhydride were obtained on the Aminco-Bowman spectrophotofluorometer with phosphorescence attachment and 150-Watt xenon source. Quartz sample tubes (I-mm id.) and a medium speed of the rotating can were used in obtaining the spectra of the adduct in an ethanol glass at 77 “K (liquid nitrogen). ( 2 ) G. H. Schenk and W. Radtke, ANAL.CHEM., 3’9,910 (1965). (3) C. A. Parker, ibid., 34, 505 (1962). (4) D. M. Hercules, “Fluorescence and Phosphorescence Analysis,” Wiley-Interscience, New York, N. Y . , 1966, pp 49, 105-7.
ANALYTICAL CHEMISTRY, VOL. 42, NO. 14, DECEMBER 1970
The ultraviolet absorption spectra of anthracene, maleic anhydride, and the Diels-Alder adduct of the two were run on both the Beckman DB spectrophotometer and on the Cary 14 spectrophotometer. On the Beckman DB, the narrow slit (manual) setting was used in recording the spectra. One-centimeter quartz cells were used in both instruments. The melting point of the anthracene-maleic anhydride adduct was determined on a Thomas-Hoover Capillary melting point apparatus. Reagents. The maleic anhydride was pulverized and stored in a sealed container. The solvents used were either reagent grade or spectral grade. The aromatic hydrocarbons were reagent grade and were used as received. The adduct of maleic anhydride and anthracene was prepared as follows : 2 grams of maleic anhydride and 2 grams of anthracene were dissolved in benzene, heated just below boiling for 2 hours, cooled, and the solid adduct was separated. It was washed twice with benzene, once with acetone, and recrystallized from acetone. The melting point was 262-3 “C(two determinations) compared to the literature value (5) of 263 “C. Procedures. Reaction times for maleic anhydride and anthracene in the absence of other hydrocarbons were studied by refluxing them in o-dichlorobenzene (bp 180’) using 50-ml conical ground glass flasks and semimicro (6inch) condensors. The presence of unreacted anthracene was checked fluorometrically. To study the measurement of pyrene, diphenylstilbene, or perylene in the presence of anthracene, stock solutions of each at 2.8 X 1W3Mwere first prepared. An aliquot of the stock solution was added to an aliquot of anthracene in a volumetric flask, such that the final solution contained 1.1 x IO-jM anthracene and 10-8 to 10-BMof one of the three above aromatic hydrocarbons. (At the lower concentrations, it was necessary to make several dilutions to reach the desired molarity.) Thus the calibration curves studied ranged from 10-8 to 10-6M. Aliquots of 10 ml of each mixture were transferred to the conical flasks, 25 + 1 mg of maleic anhydride was added, and the flasks were refluxed for 45 minutes. After cooling, the solutions were quantitatively transferred to 25-ml volumetric flasks and diluted to the mark with o-dichlorobenzene. To extract the unreacted maleic anhydride, each solution was transferred to a ground glass stoppered flask (or extraction funnel with a greaseless stopcock, such as polyethylene) and shaken with 3 ml of 0.5M sodium hydroxide for at least 15 seconds. The organic layer was drawn off, and the extraction repeated. The organic layer was then dried in a beaker by pouring it over anhydrous sodium sulfate and swirling for 30 seconds, decanting into a beaker with a second portion of anhydrous sodium sulfate, and again swirling for 30 seconds. These solutions were then read in the fluorometer, and calibration curves for each of the three individual aromatic hydrocarbons above were prepared by plotting fluorescence intensity cs. concentration. RESULTS AND DISCUSSION
Photochemistry of the Diels-Alder Adduct. After preliminary study of the Diels-Alder reaction of maleic anhydride and anthracene, the reaction appeared feasible for analytical use. A study of the photochemistry of the adduct of the above molecules was undertaken. Because the Diels-Alder adduct of TCNE and anthracene photodecomposed under irradiation at 365 nm (2), a study was first made of the ultraviolet absorption spectrum of the adduct and the stability of the adduct to ultraviolet radiation. The adduct exhibited ultraviolet absorption bands at 266 (e = 1,085) and 274 nrn (e = 1,283); a stronger band was (5) L. F. Fieser and M. Pieser, “Organic Chemistry,” 3rd edition, Reinhold, New York, N. Y., 1956, p 760.
a-2@
0
=+P
_11l1_
cr+p
0 0
@ a
a+ B
@ @
a-tl.414
x+2p
L
6+2.414
BENZENE
0.
m32p
p
A
ANTHRACENE
Figure 1. A 1-electron energy diagram for benzene (left) and anthracene (right) showing their pi molecular orbital energies as calculated by the Huckel method CY is the energy of the atomic p orbital of carbon, and p is the resonance integral constant. Theoretical energy of the lowest energy transition for benzene is 2.0 p and for anthracene 0.828 p. Because of the nature of the Huckel calculation, this is the average energy of the singlet-singlet and singlet-triplet transitions. Fortunately, the ratio of these energies in most aromatic hydrocarbons is roughly constant, making possible a rough comparison of the energies (and thus the location of the absorption bands) of the lowest energy singletsinglet electronic transitions of most aromatic hydrocarbons
observed at 214 nm but this is unconfirmed because of possible ambiguity arising from solvent absorption (the methanol cutoff is 210-220 nm). The stability of the pure adduct was tested by exciting a 10-jM solution at both 254 and 365 nrn in a filter fluorometer. Readings taken every 5 minutes for 30 minutes using a secondary filter passing radiation above 365 nm failed to indicate any fluorescence. Since anthracene exhibits intense fluorescence above this wavelength, this indicated that the adduct had not decomposed to anthracene and maleic anhydride, and that fluorescence excitation at either wavelength of other aromatic hydrocarbons in the presence of the adduct was analytically feasible. Since the above experiments indicated that the absorbed energy was not emitted as fluorescence (at least above 365 nm) and that it was not dissipated via photodecomposition, we decided to test our prediction (see above) that the adduct would be phosphorescent. Excitation at 77 OK in ethanol in a phosphorimeter indicated that it was. The phosphorescence
ANALYTICAL CHEMISTRY, VOL. 42, NO. 14, DECEMBER 1970
o
1755
WAVELENGTH.nrn.
Figure 2. Phosphorescence excitation and emission spectra for the Diels-Alder adduct of anthracene and maleic anhydride at 77 OK in ethanol
---
Excitation band, measured with the emission monochromator set at 375 nm __ Emission band, measured with the excitation monochromator set at 275 nm Spectra are uncorrected
excitation spectrum (Figure 2) indicates a lower energy excitation band (in which the 266- and 274-nm absorption bands are unresolved) and a higher energy excitation band at 225 nm,which appears to confirm the “unconfirmed” absorption band at 214 nm (6). Diels-Alder Reaction Conditions. The reaction of maleic anhydride and anthracene is quite slow at room temperature. The second order rate constant at 25.2 OC in chloroform is 0.03 X 10-3M-‘ sec-l (7). Using the second order integrated rate law with an initial maleic anhydride concentration of 10-zM,a calculation of the time for 99 reaction of anthracene indicates a time of the order of lo3hours. In o-dichlorobenzene solvent, the reaction was considerably faster at room temperature: only 48 hours was required for 75% reaction. Although the reaction is known (8) to be catalyzed by aluminum chloride, the use of this catalyst under analytical conditions produced a red-brown precipitate which prevented further investigation. The next approach investigated was that of carrying out the reaction at the boiling point (180 “C)of the o-dichlorobenzene solvent. Although the equilibrium between maIeic anhydride and aromatic dienes is less favorable at higher temperatures (9),a large excess of maleic anhydride was used lo overcome this factor. At this temperature, the reaction of lO-3M anhydride and 10-6M anthracene was indeed rapid enough for use. The reaction was followed fluorometrically after 30-, 4 5 , 60-, and 120-minute reflux times. Although a 30minute reflux usually gave good results, the results were not so reproducible as after 45 minutes’ reflux. (6) L. V. S. Hood and J. D. Winefordner, ANAL.CHEM.,38, 1922 (1966). (7) L. J. Andrews and R. M. Keefer, J. Amer. Chem. Sac., 77, 6284 (1955). (8) P. Yates and P. Eaton, ibid., 82, 4436 (1960). (9) M. C. Kloetzel, “Organic Reactions,” Vol. 4, Wiley, New York, N. Y., p 28. 1756
The next variable investigated was the maleic anhydride concentration. Although an initial concentration of 2.5 x lO-aM was used for maleic anhydride, the reaction rate was too slow and a concentration of 0.01M anhydride was adopted as the optimum concentration. Even if lO-5M anthracene were present, this would give a lo3 excess of anhydride and ensure rapid reaction. Fluorescence Quenching. When the above procedure was applied to the measurement of pyrene in the presence of anthracene, it was found that the excess maleic anhydride quenched much of the fluorescence of the pyrene. Maleic anhydride does quench pyrene’s fluorescence ( 2 ) and a 1M solution quenches anthracene effectively (10) as well. For this reason, an extraction process was investigated as a means of removing the unreacled maleic anhydride. A double extraction with O.5M sodium hydroxide was effective: in the extraction the anhydride is hydrolyzed to the sodium salt of maleic acid, leaving the Diels-Alder adduct and any other aromatic hydrocarbons behind in the organic phase. The Diels-Alder adduct has been shown to be insoluble in aqueous base during the short time required for extraction (11). Difficulties in the extraction were encountered using separatory funnels with stopcock grease. Once this type of funnel was eliminated, no fluorescent contamination from the extraction was noted. Two extractions were all that were needed to avoid quenching and permit fluorometric measurement of various aromatic hydrocarbons in the presence of anthracene. Determination of Pyrene in Anthracene. Pyrene is excited strongly at 254 nm (not shown) and only moderately in the 365-nm region (Figure 3). Using either excitation, it was possible to obtain suitable calibration curves for pyrene at both the 10-6 and lO-7Mlevels in the presence of a constant level of 10-jM anthracene. Unfortunately, the calibration curves obtained using excitation at 254 nm both pass above the origin by 5 to 10 units, apparently because the DielsAlder equilibrium is not favorable enough to convert all of the anthracene to the adduct. A different calibration curve would have to be prepared using less anthracene if the sample contains very low levels (1O-gM) of anthracene. For the same reason, it was not possible with a lO-+M level of anthracene to measure 10-8M levels of pyrene accurately at 254 or 365 nm. Since anthracene is excited at either wavelength (Figure 3), a trace of it will interfere when 10-*M levels of pyrene are excited. If less anthracene were present, it is clear from equilibrium principles that there would be less interference. It was our aim to test conditions where a relatively large concentration of anthracene might be present, however. The results for the determination of pyrene in the presence of anthracene after the Diels-Alder reaction are given in Table I. Since the excitation of pyrene at 365 nm is only moderate, it was necessary to use a 10X mercury source intensity setting on the Turner fluorometer. The 405-nm sharp cut secondary filter passed nearly all of the intense emission bands of pyrene (Figure 3) and permitted analysis at the lG-*M level. The results are about comparable to those obtained by excitation at 254 nm. Since the 254-nm excitation is more efficient, it was possible to use a 394-nm interference filter as secondary filter to pass the intense pyrene emission in the 385-405 nrn region. This still permitted analysis at the lO-7Mregion. (10) J. P. Simons, Tmrzs. Faraday Sac., 56, 391 (1960). (11) I. Ubaldini, V. Crespi, and F. Guerrieri, AH/?.Chim Appl., 39,77 (1949).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 14, DECEMBER 1970
I
.9
>
EMISSION
t v)
z
w I-
ABSORPTION
.7
z -s w
r c
.5
a .4 -I
w
a
.3 .2
I
W A V E L E N G T H , nrn.
Figure 3. Fluorescence excitation (right) and emission spectra (left) of anthracene and pyrene
---
Represents respective bands for pyrene
- Represents the respective bands for anthracene
Spectra are uncorrected Inner Filter Effect. The possible interference of the adduct via the inner filter effect on the determination of pyrene was also investigated. The basis for the inner filter effect referred to here is the absorption of a significant portion of the exciting radiation in the front of the cell, such that the solution at the back of the cell is exposed to a lower intensity excitation (12). In this case, the adduct absorbs enough exciting radiation that the fluorescence of the pyrene is diminished at the back of the cell. Since the Diels-Alder adduct has a broad band at 264 nm but does not absorb in the 365-nm region, it can only cause an inner filter effect when 254-nm excitation is used. To test for the inner filter effect, solutions of pyrene alone were read on the fluorometer, a small volume of the DielsAlder adduct was added, and the fluorescence was read again. (The fluorescence readings were corrected for volume changes.) The results are shown in Table 11. Even at concentrations above the lO-5M level of anthracene used in the study, the inner filter effect is absent. However, when the adduct concentration is increased to the 10-4M level, a definite decrease in the fluorescence of pyrene is observed. Determination of Diphenylstilbene and Perylene in Anthracene. The trans-p-p’-diphenylstilbene (DPS) was excited efficiently at 365 nm so that 254-nm excitation was not needed. The 394-nm interference filter appeared to be more sensitive than the 405-nm narrow pass filter as a second filter. A linear calibration curve was obtained from 8.9 x 10-8M to 1.1 X 10-6M DPS using either secondary filter. Some analytical work was done at higher concentrations, but these samples had to be diluted for on-scale readings. The DPS stock solutions had to be dissolved in benzene, but the amount of benzene present after the final dilutions was too small to affect calibration curves via the inner filter effect, etc. Before discussing the analysis of perylene-anthracene mixtures, it should be pointed out that perylene can be (12) C . A. Parker, “Photoluminescence of Solutions,” Elsevier, Amsterdam, 1968, pp 20, 222.
Table I. Determination of Pyrene in the Presence of 1.X x lO”M Anthracene Pyrene found, M Pyrene present, XEX = 365 nma XEX = 254 nmb M XE>l = 405 nrn XEM = 390 nrn 2.8 5.6 11.2 2.8 5.6 11.2
x
x
x x x
x
x x x
2 . 6 x 10-5 6.0 x 10-5 11.7 X 10-6
10-5 10-5 10-7 10-7 10-7
2.8 5.8 11.3 10-6 2.6 x 10-7 6.1 X 10.8 10-7
...
...
x
...
Intensity setting: 1OX on Turner mercury source. b Intensity setting: 1X on Turner mercury source for 10-BM pyrene and 3 X for 10-6M pyrene. a
Table 11. Evaluation of Inner Filter Effect of Diels-Alder Adduct on Determination of Pyrene [Excitation at 254 nm (3X intensity); 394 nm interference secondary filter] F, Turner units 4
Pyrene present, M 1.1 x 2.25 x 3.37 x 4.50 x 5.62 X
No adduct
10-6 10-5 1010-6
10-6
’
20 45 68 74 93
x
10-5~4
adduct 20
45 68 74 95
x
10-4~
adduct 16 40 63 68 85
excited at 405 nm (485-nm secondary filter) without exciting anthracene. However, by using the Diels-Alder reaction to convert anthracene to the adduct, it becomes possible to excite perylene more eficiently at 365 nm. Using this excitation, a sharp cut secondary filter which passes all fluorescence emission beyond 41 5 nm could also be used. Since the fluorescence emission of perylene begins at 430 nm, this combination of excitation and emission filters permitted a more sensitive measurement of perylene than excitation at 405 nm. Excitation of perylene at 254 nm was also used and
ANALYTICAL CHEMISTRY, VOL. 42, NO. 14, DECEMBER 1970
1757
Table 111. Determination of Diphenylstilbene (DPS) or Perylene in the Presence of 1.1 x lO+M Anthracene DPS found, A4___XEX = 365 nm XEX = 365 nm DPS present, M XEAI = 405 nm XEU = 390 nm 8.9 x 8.9 x 10-8 8.9 x 2.0 x 10-7 1.8 x 10-7 1.9 x 10-7
2.8 x 10-7
Perylene present, M 4.5 x 10-8 1 . 1 x 10-7 2.2 x 10-7
2.9 x 10-7 2.8 x 10-7 Pervlene found. M XEX = 365 nm XEX = 254 nm XEN = 415 nm XEM = 415 nm 4 . 0 X 10-8 4 . 5 x 10-8 1.2 x 10-7 1 . 3 X 10-7 2.3 X 2.3 X 10-7
Table IV. Determination of Anthracene by Difference in 5 X 10-5M Pyrene and 1.2 x 10-6M D P S
Anthracene M Present 2.9 x 10-5
Found 3.2 x 10-5
8.6 X
8.4
x
10-5
Difference in fluorescence intensitiesa 11.5 30.5
11.5 X 11.9 X l(9-5 41.5 14.4 x 10-5 14.2 x 10-6 51 5 The fluorescence intensity was 23.5 Turner units after DielsAlder reaction of the anthracene in each sample.
was found to be more efficient than at 365 nm. Using 254 nm, a linear calibration curve was obtained from 1 x loF8 to 5.6 X lO-’M perylene. Using 365-nm excitation, a linear to above calibration curve was obtained from 4.5 X 6 x lO-7M. See Table I11 for analytical results. The calibration curves for both DPS and perylene generally passed through the origin. Only in the case of measurement of DPS using the 394-nm secondary interference filter did the curve pass about 3 units above the origin. Determination of Anthracene by Difference. Determination of anthracene by difference appeared to be possible by measurement of the total fluorescence of a sample before adding maleic anhydride, followed by measurement of the residual fluorescence after the Diels-Alder reaction of ahthracene.
1758
ANALYTICAL CHEMISTRY, VOL. 42,
As in the regular procedure, 10-ml aliquots of mixtures of anthracene, pyrene, and DPS were treated with maleic anhydride, extracted, and diluted to a final volume of 25 ml. Untreated 10-ml aliquots were also diluted to a final volume of 25 ml. The fluorescence of all samples was measured by exciting at 365 nm and using the 394-nm secondary interference filter. To prepare a calibration curve, the difference between the two fluorescence readings for a given anthracene concentration was plotted against the molarity of anthracene. The calibration curve obtained was linear over the region from 1.4 X 10-5 to 1.7 X lO-4M anthracene. The results of analysis for anthracene are shown in Table IV. To prepare an accurate calibration curve for a complex mixture would probably be impossible, but for a mixture of 3 or 4 aromatics it should not be too difficult. However, the fluorescence intensity of the sample after the reaction of anthracene would have to be known before standards could be prepared that would contain a total amount of the other aromatics that would give the same fluorescence intensity as the sample. Potential Applications. One possible practical application of this study would be in the fluorometric measurement of chromatographic fractions containing anthracene plus one or more other fluorescent species. For example, Commins (13) found that anthracene occurred in a pyrene-fluoranthene fraction of an alumina chromatographic separation of aromatic hydrocarbons in “atmospheric soot.” It should be possible to convert the anthracene to the Diels-Alder adduct by our procedure, and measure the pyrene fluorometrically without interferenct from the anthracene. Since maleic anhydride undergoes the Diels-Alder reaction with other conjugated molecules such as naphthacene, pentacene, and 1,2-benzanthracene, possibly this procedure could be applied to fluorometric measurement of mixtures containing these molecules after converting them to nonfluorescent or weakly fluorescent adducts of maleic anhydride. RECEIVED for review July 27, 1970. Accepted September 14, 1970.
(13) B. T. Commins, Analyst, 83, 386 (1958).
NO. 14, DECEMBER 1970
