in the presence of P-methylstyrene (BMS). The spectra of the samples in methanol and acidified methanol are shown in Figure 4. The difference in absorbance a t the analytical wavelength was used for the quantitative measurement. The spectrum obtained by differential spectrophotometry is shown in Figure 5. The results obtained by both methods of spectrophotonietry are given in Table IT’. A cinnamaldehyde-8methylstyrene sample was analyzed on six different occasions to obtain an indication of the reproducibility of the method (Table V). Furfural (FUR) was analyzed in the presence of dimethylphthalate (DMP) using the same methods as were employed for cinnamaldehyde. The results obtained for three synthetic samples are shown in Table VI. The data obtained for both the cinnanialdehyde and furfural analyses demonstrate the applicability of the aldehyde-methanol reaction in the analysis of aldehydes by ultraviolet spectrophotometry. The precision of both methods of spectrophotometry was con~parable and both standard deviations were of the same magnitude as those obtained by conventional tech-
Table VI.
25.2 25.2 25.2
1.487 4.461 7.438
Determination of Furfural
93.5 100.5 100.1
1.390 4.483 7.442
1.395 i.448 i ,437
93.8 99.7 100.0
niques with monocomponent systems. Recoveries mere also within acceptable limits. The interferences and exceptions discussed in connection with the qualitative aspects of this technique also apply to quantitative analysis. A subtle application of this technique of ultraviolet spectrophotometry to both qualitative and quantitative analysis is the utilization of the acetal reaction to alter aldehyde absorption in the study of other ultraviolet-absorbing compounds.
Inc., for granting permission to publish this work.
ACKNOWLEDGMENT
RECEIVEDfor review June 25. 1962. Accepted November 20, 1962. Division of Analytical Chemistry, 142nd Meeting, ACS, Atlantic City, N. J., September 1962.
The authors express appreciation to Ernest TV. Robb for his helpful suggestions. They also thank Philip Morris,
LITERATURE CITED
(1) Djerassi, Carl, “Optical Rotatory Dispersion,” Chapter 11, McGraw-Hill, .New York, 1960. (21 . . Forrester, J. S., ANAL. CHEM.32, 1668 (i96oj. (31 Kulka. K.. Am. Perfumer Essent. Oil Rev. 54,’136i1949). (4) Melchior, N.,J. Am. Chem. SOC.71, 3651 (1949). (5) Wheeler, 0. H., Msteos, J. L., ANAL. CHEM.29,538 (1957). ~
Spectrophotofluorometric Studies of Some Aromatic Aldehydes and Their Acetals EDWIN P. CROWELL’ and CHARLES
J.
VARSEL
Philip Morris Research Center, Richmond, Vu.
b The fluorescence quenching effect of the carboxaldehyde group on aromatic systems substituted with hydroxy, alkoxy, and cyano groups, and polycyclic aromatic aldehydes has been investigated to determine if the quenching effect of the carbonyl could be overcome by the fluorescentpromoting properties of the additional fluorophoric groups. In methanol, nine of the 26 aldehydes studied fluoresced. The fluorescence of only five of these compounds was attributed to the aldehyde species. All or part of the emission of seven of these aldehydes resulted from the methyl hemiacetal of the aldehyde in the alcohol solvent. The acetal reaction was a convenient and useful analytical tool for the study of fluorophorically-substituted aromatic aldehydes. Fluorescence ftom normally nonfluorescent-substituted aldehydes was observed when methanol solutions of these aldehydes were acidified. This fluorescence resulted because the quenching effect of the aldehyde group was eliminated as a result of acetal formation.
S
is a relatively new technique in the field of analytical chemistry. As a result, much exploratory work is needed on new techniques and compilation of reference spectra to further the application of spectrofluorescence to chemical analysis. No investigations have been reported on the fluorophoric influence of the carbonyl group in aromatic systems. Duggan and coworkers ( 2 ) and Williams (4) in their discussions concluded that to induce fluorescence into the simple aromatic nucleus, a t least one electrondonating group must be present. From this generalization it can be expected that the carbonyl group would not be a fluorophore and, further, as part of a complex system would probably exert a quenching effect. The objective of this present study was twofold: to obtain an indication of the relative fluorescence quenching or inhibiting strength of the aldehyde group, and to investigate the acetal reaction in conjunction with spectrophotofluorescence as a technique for the PECTROPHOTOFLUOROSIETRY
characterization of substituted aromatic aldehydes. Our observations indicated that aromatic aldehydes generally do not exhibit fluorescence a t room temperature with presently available commercial instruments. It was therefore thought desirable to study the possibility of employing a reaction that would mask the quenching influence of the aldehyde group and permit a spectrophotofluorescence study of the remaining fluorophoric structure. It naturally followed that for this technique to be applicable to the study of aromatic aldehydes, these aldehydes must possess, in addition to the aldehyde group, other groups that will promote fluorescence in the aromatic nucleus. Aromatic aldehydes substituted with hydroxy, alkoxy, cyano, alkenyl, or amino groups or polycyclic aromatic aldehydes are compounds suitable for a study of this type. Acetal formation in conjunction with 1 Present addrese, American Cyanamid Co., P. 0. Box 400, Princeton, N. J.
VOL. 35, NO. 2, FEBRUARY 1963
189
Table I.
Summary of Fluorescence Properties
Methanol Ex.mx. Em.,,,. (mp) none none 280 310 none none 282 319 340 445 278 460 358 263
+ acid
~
Compound o-Methoxybenzaldehyde p-Methoxybenzaldehyde m-Methoxybenzaldehyde 2,4-Dimethoxybenzaldehyde 3,4-Dimethoxybenzaldehyde 3,5-Dimethoxybenzaldehyde
2,5-Dimethoxybenzaldehyde 2,3-Dimethoxybenzaldehyde
4-Ethoxy-3-methoxybenzaldehyde 3,4,5-Trimethoxybenzaldehyde Piperonal o-Hydroxybenzaldehyde p-Hydroxybenzsldehyde m-Hydroxybenzaldehyde
338 270 283
2,4-Dihydroxybenzaldehyde
3,4-Dihydroxybenzaldehyde 2,5-Dihydroxybenxnldehyde
362 261
4-Hydroxy-3-methoxybenzaldehyde 4-Hydroxy-3-ethoxybenzaldehyde 2,4.6-Trihydroxybenzaldehyde 4-Hydroxy-3,5-dimethoxybenzaldehyde Cinnamaldehyde
2-Xaphthaldehyde
Phenanthrene-9-aldehyde 9-Anthraldehyde
p-Cyanobenzaldehyde
Z
=
Implication
0.01 0.01 0.01
~
KQ 0.10 0.05 0.11 0.06 0.11 0.09
0.48
298
327
0.26
465
0.01
280
324
0.02
320 none none none none none none none none 485
0.01
0.03
Solvent
Ether HzO
0.01
480
0.09
500
0.01
Ether HzO
none 375
495
0.01
none 3.54 300 264 none 328 260 none 270 413 380 none
Ether H?O
Ether
289 280 281 283 285 284 302
0.10
320 none 324 313 312 314 325 322 335
0.42 0.12 0.05 0 11
0.01 0.14 0.30
285 284
322 322 none none
0.14 0.14
none
260 383 2951 280
316
0.51
332
0.12
Ether Hz0
255 296
364 349
0.51
Ether
256 349 365 385 239 279
406 426
1.96
Ether Hz0
312
0.04
Ether
255 296 278 256 349 365 385 239 279
364 349 380 406 429 495
0.07
312
0.03
0.09
KQ
465
Hz0
282
Other EX.,sx. (mp)
none 335 285 none 360 268 none 345 275
none none none none
0.09
EXPERIMENTAL
ANALYTICAL CHEMISTRY
KQ
332
The fluorescent spectral properties were determined with an Aminco-Kier Spectrophosphorimeter adapted for room-temperature fluorometric measurements. The source employed with this equipment was a high pressure Osram xenon arc lamp. A lP21 phototube, RCA 9-stage type with S4 response, was used as the detector. A 1.0 mm. wide shutter was used at the phototube, and the exciting beam as well as the emitted beam each were passed through a series of slit widths of
Methanol . Em.,,,. (mp) (mp) 311 282 312 276 310 280 316 282 318 281 320 282
Ex.IZlax
280
spectrophotofluorescence is a convenient and useful analytical tool for the study of fluorophorically-substituted aromatic aldehydes. Fluorescence from these normally nonfluorescent substituted aldehydes resulted when methanol solutions of these aldehydes were acidified. This fluorescence resulted because the quenching effect of the aldehyde group was eliminated as a result of the acidcatalyzed reaction between the aldehyde and the methanol t o form the corresponding acetal.
190
~~
HzO
3, 1, and 2 mm., respectively (with the 1-mm. defining slit the band pass is equal to 4 mp). The emission monochromator was calibrated with a quartz Pen-Ray cold cathode tube containing a n argon-mercury filling. The excitation monochromator was calibrated by observing the coherent light scatter from an aqueous polystyrene suspension. The recorded fluorescence intensities were related to the fluorescence observed at 450 mp for a quinine sulfate solution, 1 pg. per ml. in 0.1N H2S04, excited at 350 mp. The fluorescence intensity of the sample in arbitrary instrument units was divided, first by the sample concentration in micrograms per milliliter, then by the fluorescence intensity of the quinine sulfate standard. The value thus obtained, KQ, is the fluorescence intensity of the compound relative to quinine sulfate. The reciprocal of this constant is the concentration of the compound, in micrograms per milliliter, that will give the same fluorescence intensity as 1 pg. per ml. of quinine sulfate. The fluorescence properties of the compounds studied were determined initially in methanol. The solution x i s
445
470 515
then acidified with one drop of concentrated sulfuric acid per 10 ml., and the fluorescence properties of the corresponding acetal were determined. The compounds used in this study were obtained from Aldrich Chemical Co. Melting points were checked as a n index of purity. Khere necessary the compounds were recrystallized from benzene. RESULTS A N D DISCUSSION
The experimental data determined for the aromatic aldehydes investigated in this study are listed in Table I. All of the aromatic aldehydes included in this study contained, in addition t o the carbonyl group, substituents which normally enhance the fluorescence of the benzene nucleus to the extent that the emitted fluorescence can be detected with commercially available equipment. The hydroxy, alkoxy, alkenyl, and cyano groups, and polycyclics were investigated as substituents in aromatic aldehyde systems.
EXCITATION
EMISSION
Table II. Compounds That Ftuoresced in Methanol
I
Hemi- Aldeacetal hyde m-hlethoxybenzaldehyde X 3,5-Dimethoxybenzaldehyde X 2,3-Dimethoxybenzaldehyde X 2,5-Dimethoxybenzaldehyde 2,5-Dihydroxpbenzaldehyde 9-An thraldehyde Phenanthrene-9-aldehyde 2-Naphthaldehpde p-Cyanobenzaldehyde a Observed in water only x - Species fluorescing
WAVELENGTH - m y
Figure 1 . Excitation and emission spectra of substituted benzaldehydes in methanol A.
E.
m-Methoxybenzaldehyde 3,5-Dimethoxybenraldehyde: (. .) Excitation spectrum for 31 9-mp emission peak (-) Excitation spectrum for 445 -mp emission p e a k 2,5-Dimethoxybenzaldehyde
..
--
C.
Generally, the sub5tituted aromatic aldehydes investigated did not fluoresce. This indicates that the aldehyde carbonyl group exerts a strong quenching effect on the aromatic system. Of the 26 aldehydes studied, fluorescence was obrerved from the methanol solution of only the nine compounds listed in Table 11. \t7ith fire of these compounds, fluorescence was attributed to the aldehyde species. I n seven of the cases where fluorescence was detected in methanol all or part of the emission resulted from the methyl hemiacetal of t h e aldehyde. Figure 1 shows that excitation arid emission spectra of three methoxysubstituted benzaldehydes in methanol. nz-Nethoxybenaaldehyde exhibited the emission spectrum of its hemiacetal. The emission spectrum of 3,s-dimethoxybenzaldehyde in methanol exhibited characteristics of both the hemiacetal and aldehyde. The two fluorescence peaks each had different excitation spectra indicating that two species were present in the system. The fluorescence observed with 2,s-dirnethoxybenzaldehyde was only from the aldehyde species. The conclusion as t o whether the observed fluorescence resulted from the aldehyde or its hemiacetal was deduced from the following considerations: If the emission and excitation spectra observed in methanol and acidified
methanol Ivere the same except for relative intensities i t was concluded that the species fluorescing was the hemiacetal. When this similarity exists, the molecular structures in both instances must possess the same fluorophoric configuration which is the case with the hemiacetal and acetal of the same aldehyde. This similarity was exemplified by the three polycyclic aldehydes studied vhich exhibited the same emission and excitation spectra in both solvent systems. The excitation spectra also resembled the ultraviolet absorption spectra of the acetals rather than the aldehydes. The relative intensity was always greater in the acid system. The 495-mp peak in 9-anthraIdehg.de which was very weak was due t o the aldehyde species. It was absent in the acidified methanol spectrum. The compounds that fluoresced in methanol were analyzed in water.
Table 111.
2,3-
2,42,5-
394-
X X X X
x
x
X
Xa X"
x
X
Here, where hemiacetal formation is not possible, only fluorescence of the aldehyde resulted. The excitation spectra obtained in the mater system resembled the ultraviolet absorption spect r a of the aldehydes. The aldehyde fluorescence of 2-naphthaldehyde and phenanthrene-9-aldehyde in methanol \vas too feeble in comparison to the hemiacetal fluorescence t o be detected, but was clearly evident in the water solution. V i t h regard t o the measurement of aldehyde fluorescence in solvents other than alcohol, initially ether was employed but aldehyde fluorescence could not be detected in this solvent. JTilliams ( d ) , in his work on hydroxybenzoic acids, reported that the nonionized forms of the monohydroxy benzoic acids did not fluoresce. This wm also the case with the monohydroxy benzaldehydes investigated in this work. according t o Williams, the dihydroxybenzoic acids did fluoresce. He reported descriptive data on the fluorescence intensities of these dihydroxybenzoic acids. From a comparison of our corresponding data on benzaldehydes with those reported for the dihydroxy benzoic acids (Table III), it is erident that the carboxaldehyde group has a stronger quenching effect on the aromatic nucleus than does the carboxylic acid group. Further, it appears that in the benzaldehyde series, the probability of radiative transition from the excited singlet t o ground singlet states is greater for the alko~\--substituted system than for the hydroxysubstituted system. This is indicated from a comparison of the relative
Comparison of Relative Intensities of Dioxy-Benzaldehydes and Benzoic Acids
Dihydroxybenzoic acids weak medium strong weak
Dihydroxybenzaldehydes, KQ
...
none 0.03
none
Dimethoxybenzaldehydes, KQ 0.01
none 0.48
none
VOL. 35, NO. 2, FEBRUARY 1963
0
-_191
~
Table IV.
~~~~
Comparison between Fluorescence Properties of Acetals and Their Corresponding Alkyl Derivatives Acetals Alkyl derivatives
Ex.max. (md 280 28 1 283 260 285 295 279 239 280 256 349 365 385
Compound o-Hydroxybenzaldehyde
p-hydroxy benzaldehyde
m-Hydroxy benzaldehyde Cinnamaldehyde p-Cyanobenzaldehyde 2-Naphthaldehyde 8-Anthraldehyde
Table V. Intensities of Acetals Disubstituted Benzaldehydes
2,4-Dimethoxy 2,PDihydroxy 2,5-Dimethoxy 2,5-Dihydroxy 3,4-Dimethoxy 3-Methoxy-4-ethoxy 3,PDihydroxy 3-Methoxy-4-hydroxy 3-Ethoxy-4-hydroxy Piperonal
Ex.,,. (mr) 313 312 314 316
0.05
0.11 0.51
Compound o-Cresol p-Cresol m-Cresol Methylstyrene
312
0.03
p-Tolunitrile
332 406 426
0.12 1.96 0.92
2-Methylnaphthalene 9-Methylanthracene ( 3 )
KQ
0.12
Examax. (mr) 279 283 279 260 283 295 272 240 280 252 343 362 382
Em.max.
KQ
314 316 313 320
0.13 0.19 0.11 0.64
312
0.05
335 410 434
0.28 2.05 0.94
6.
A
of
KQ 0.06 0.01 0 26 0 30 0.11 0.10
0.14
0 14
0 14 0.42
intensities of the fluorescence of 2,5dimethoxy- and 2, j-dihydroxybenzaldehydes. The dimethyl acetals, formed by acidification of the methanol solutions, of most of the aldehydes included in this study were fluorescent. The acetal group is totally inactive as a fluorophore in the ultraviolet and visible regions of the electromagnetic spectrum. This group, therefore, will have a n effect similar to a n alkyl group when considered as a part of a fluorophoric system. The data in Table IV compare the fluorescence properties of some of the acetals with their corresponding alkyl derivatives. The fluorescence properties of the acetals of the hydroxybenzaldehydes are similar to the cresols; the acetal of cinnamaldeliyde resembles p-methylstyrene, etc. Even more striking are the similarities between the multipeak spectra of the acetal of 9-anthraldehyde and 9-methylanthracene. The excitation spectra of the acetals resemble the ultraviolet spectra of the corresponding molecular nucleus without the carbonyl group. The clearest example of this effect is observed by the comparison of the excitation spectrum of the acetal of 9-anthraldehyde with the ultraviolet spectrum of anthracene as shown in Figure 2. A comparison of the relative fluorescence intensities of the acetals of the mono-substituted benzaldehydes shows that there is apparently no difference
192
~~~
ANALYTICAL CHEMISTRY
2
300
400
300
WAVELENGTH
Figure 2. Comparison absorption spectra
-r
400 n ~
of excitation and
ultraviolet
.
A.
(. .) Ultraviolet absorption spectrum of anthracene (-1 Fluorescence excitation spectrum of acetal of 9-anthraldehyde
B.
Fluorescence emission spectrum of acetal of 9-anihraldehyde
produced between the methoxyl and hydroxyl groups, but there is a difference resulting from the position of the substituent relative to the acetal group. With t h e acetals of the disubstituted benzaldehydes (Table V), again there appeared to be no difference in relative intensities between methosyl and hpdroxyl derivatives, but there were sharp differences resulting among position isomers. The lower relative intensities measured for the two 2,4-isomers were accounted for by the fact that the acetal reaction is incomplete with these compounds. Ultraviolet absorption experiments have shown that the 2,4dimethosybenzaldehyde reaction is 65% complete and the 2,4-dihydrosybenzaldehyde reaction is 17Yo complete. The acetal of piperonal exhibited a relative intensity considerably higher than the intensities observed for the other 3,4-dioxy-benzaldehydes that were studied. Increased fluorescence intensity resulted because piperonal is
more reactive toward addition reactions than the 3,4-diosy derivatives. As a result there was less free aldehyde available in the sample to quench the fluorescence emission of the acetal of piperonal. S o fluorescence was observed with the acetals of the trioxy derivatives of benzaldehydes. This is in agreement with literature (1) data which report that trihydrosybenzenes do not fluoresce. LITERATURE CITED
(1) American
Instrument Co., Silver Spring, Md., Bull. 2278, 1959. (2) Duggan, D. E., Bowman, R. L., Brodie, B. B., Udenfriend, S., Arch. Biochem. Biophys. 68, 1 (1957). (3) Safficki, E., Intern. J. Air Pollution 2 , 253 (1960). (4) Williams, R. T., J . Roy. Inst. Chenz. 83, 611 (1959,). RECEIVEDfor review August 24, 1962. Accepted November 20, 1962. Division of Analytical Chemistry, 142nd Meeting, ACS, Atlantic City, N. J.
