smaller g value for the lignite radical as compared to that of the peat indicates that a larger polynuclear condensed ringsystem structure is present in the basic semiquinone structure. During further metamorphosis these rings become larger and thus the free radical electron spends less time on the oxygen part of the semiquinone and more time on the hydrocarbon portion. The g values are, at least for the lower rank bituminous coals, too high to be accounted for by pure hydrocarbon type radicals. The formation of more radicals, probably involving oxygenated species, accounts for the increase in the number of unpaired electrons with increasing coal rank. For the higher rank bituminous coals and the
anthracitic coals, with the exception of the meta-anthracite, it is difficult to say whether the ESR signal is caused by a very large aromatic semiquinone radical in which the unpaired electrons are largely delocalized over the ring and thus spend little time on the oxygen atom, or the free radical is truly hydrocarbon in structure. Electrical conductivity effects obscure the interpretation of the spectrum of the very high rank meta-anthracite. RECEIVED for review January 24, 1968. Accepted June 20, 1968. Paper presented in part at the 1967 Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy.
Fluorometric Determination of Acrolein and Related Compounds with mAminophenol R. A. Alarcon The Children’s Cancer Research Foundation, and The Department of Pathology, Harvard Medical School, Boston, Mass. 02115
A fluorometric method has been developed to measure minimal amounts of acrolein and related compounds through their reaction with m-aminophenol in an acid media. Concentrations of acrolein as low as 5 X pmole/ml can be determined by this fast and simple procedure. The fluorescence spectra of the acrolein product at neutral pH (A excit. max. 400 mp; X fl. max. 510 mM), were shown to be unique among the many spectra from the numerous compounds tested and appear, so far, specific for the acrolein derivative. The fluorescent compound from acrolein has been identified as 7-hydroxyquinoline by the excitation and fluorescence spectra in any given solvent or pH value, by the identity of the UV spectra, the R, values in paper chromatograms, sublimation characteristics, and similarity of the minimal concentration ranges that could be measured fluorometrically.
A FLUOROMETRIC METHOD has been developed to measure minimal concentrations of acrolein and related compounds, well below the levels reached with other procedures. A reaction involving resorcinol and acrolein that yields a fluorescent product has previously been communicated by us ( I ) . This reaction is highly specific, but lacks a significant increase in sensitivity if compared with some of the methods most widely used, such as the colorimetric procedures with color-producing reagents tryptophan (Z), phloroglucinol (3), or 4-hexylresorcinol (4). A project to study the role of acrolein in biochemical reactions created the need for a more sensitive detecting procedure. Acrolein has been recently isolated in these laboratories and from some enzymically oxidized biogenic amines (3, theories that this aldehyde or closely related compounds might play a role in vivo have already been reported (6). Consequently, experimentation to develop a more sensitive method of detection for this aldehyde was undertaken. Exploratory studies with compounds related to resorcinol, (1) R. A. Alarcon, Arch. Biochem. Biophys., 113,281 (1966).
(2) S. J. Circle, L. Stone, and C. S. Boruff, IND. ENG. CHEM., ANAL.ED., 17,259 (1945). (3) W. C. Powick, Ind. Eng. Chem., 15, 66 (1923). (4) I. Cohen and A. P. Altshuller, ANAL.CHEM., 33,726 (1961). (5) R. A. Alarcon, Arch. Biochem. Biophys., 106,240 (1964). (6) M. Soriano, Med. Clin. ( B u r c e h a , Spain), 38, 259 (1962).
1704 *
ANALYTICAL CHEMISTRY
as well as with monophenol and derivatives, showed that a simple and highly sensitive fluorometric procedure for acrolein and related compounds could be developed, with m-aminophenol in acid solution as reagent. EXPERIMENTAL
Instruments. A Farrand filter fluorometer (Model A) was used for most of the routine determination and comparison of fluorescence values. Calibrated borosilicate glass round cuvettes 75 X 10 mm (outside diameter) held the samples. The filter combination consisted of a Corning 9863 (254-420 mp) as the primary filter, and a Wratten 65-A (peak 495 mp) secondary filter arrangement. Slit 6 was used in all readings, The complete excitation and fluorescence spectra, and some quantitation when indicated, were obtained with the AmincoBowman Spectrophotofluorometer (American Instrument Co., Silver Spring, Md.). Samples were contained in 12 x 12 x 48 mm (outside dimensions) fused quartz cells, The same slit arrangement (No. 3) was used throughout the work. Reagents. All compounds used were of the highest available purity. MIXEDREAGENT.250 mg of m-aminophenol plus 300 mg of hydroxylamine hydrochloride were dissolved in 25 ml N HC1. When specified, m-aminophenol alone in acid solution was also used. These reagents could be stored at room temperature 3 or 4 days if protected from direct light. Solutions. Acrolein standard solutions, as well as solutions of aldehydes and most of the other compounds studied, were prepared with distilled water or ethanol immediately before their use. Procedure. The compounds to be tested were used in concentrations up to an arbitrary limit of 1 pmole/ml, which was equivalent to a thousand-fold the concentration of acrolein most frequently used as fluorescence standard throughout this work. If for any given compound, no significant fluorescence was observed at this maximal concentration with a fluorometer sensitivity setting adequate to above standardi.e., Blank = 0, Acrolein 0.001 pmole/ml = 25 or 30-it was considered as giving a negative result and no higher concentrations were explored. COMPOUNDS IN AQUEOUS SOLUTION.To 2 ml of an aqueous solution containing the compound to be tested for fluorescence, were added 0.5 ml of the mixed reagent and 0.5 ml of 5N HC1. Blanks were similarly prepared. The re-
sulting mixtures were heated in a boiling water bath for 10 minutes, and subsequently cooled in tap water. Fluorescence readings at room temperature were usually made immediately afterward. However, the elapsed time after the heating period is not critical as the fluorescence is stable for several hours. COMPOUNDS IN ALCOHOLICSOLUTION. Alternatively, similar fluorescence values as those given by aqueous solutions were also obtained for acrolein and crotonaldehyde when dissolved in 95W ethanol. In this case, after the addition of the acid and the reagent to the 2 ml of the ethanolic solution, the mixtures were heated in a water bath at 65 "C for 45 minutes. Blanks with ethanol, required for the "Zero" reference point of the galvanometer readings, were similarly prepared for each assay. RESULTS AND DISCUSSION
Several compounds, most of them aldehydes, were found to produce fluorescences when heated with an acid solution of rn-aminophenol. In equimolar amounts, acrolein produced the strongest fluorescence followed by crotonaldehyde, these fluorescences being far more intense than those of any other compounds tested (Table I). As expected, compounds known to be capable to generate acrolein either by direct hydrolysis (7) (such as the four compounds following acrolein in Table I), or after their enzymatic oxidation [as already demonstrated for some biogenic amines (5)], also gave intense fluorescences (included in the first section of Table I). With the incorporation of hydroxylamine hydrochloride to the reagent solution, a further increase in the differences among the fluorescence intensities produced by acrolein and crotonaldehyde on one hand, and the fluorescences from most of the rest of the compounds studied on the other, was obtained. (7) H.L.Yale and J. Bernstein, J. Am. Chem. SOC.,70,254(1948).
Negative results as previously defined, under those experimental conditions, were observed with the following alcohols, amines, polyamines, amino acids, and other compounds tested : methanol, ethanol, propanol, glycerol, acetone, acrylonitrile, propylamine, heptylamine, 1,2-propanediamine, 1,3-propanediamine, putrescine, cadaverine, 3,3-diaminodipropylamine, spermine, spermidine, glycine, phenylalanine, lysine, arginine, cysteine, methionine, 4-aminobutyric acid, malic acid, and sodium ascorbate. Effect of Time and Temperature upon Reaction. In studies made with aqueous solutions of acrolein, roughly 80 of the possible maximal fluorescence was obtained in the first 10 minutes of heating the reaction mixture in the boiling water bath (Figure 1). The fluorescence, thereafter, increased very slowly, reaching a maximum about 20 minutes later. No further increase could be observed even after 60 minutes of heating. The effects of other temperatures on the rate of production of fluorescence are also recorded (Figure 1). Variations on Acidic Media. The hydrochloric acid concentration in the reaction mixture with acrolein was varied from 0.1N to 4N and the resulting fluorescence intensities were compared. The highest fluorescences were recorded when the acid concentration was in a range from 0.5Nto 1N. Fluorescences could also be obtained by replacing the hydrochloric acid with other acids. Phosphoric and acetic acid under the same experimental conditions, yielded 10-2OW less fluorescence, respectively, while sulfuric acid increased it around 10%. Nevertheless, for technical reasons as well as to avoid some secondary effects of sulfuric acid, hydrochloric acid was used exclusively. Effect of Variations in Concentration of Reagents. A constant concentration of hydroxylamine hydrochloride (3.5 x mmole/ml) was maintained in the reaction mix-
Table I. Fluorescences Obtained with Several Compounds upon Their Reaction with rn-Aminophenol-Hydroxylamine Reagent Relative fluorescence Concentration values," Compound in solution pmole/ml X fl. = 495 mp X excit. max.,b mp X fl. max.,b mp 30 Acrolein 0.001 350-5 505-10 505-10 Diethylacetalc 0.001 350-5 38 0.001 Hydrate diacetatec 505-10 350-5 48 P-Ethoxypropionaldehyde 0.001 diethylacetap 350-5 505-10 27 P-Methoxypropionaldehyde
0.001
26
350-5
505-10
dimethylacetalc Oxidized spermine 0.00Y 350-5 27 500-5 O.OOSd Spermidine 350-5 13 500-5 O.OOSd Allylamine 350-5 505-5 19 Crotonaldehyde 0.001 20 345 495 3-Propylacroleinc 0.01 350 20 500 Sorbic Aldehydec 0.1 15 355 500 Acetaldehyde 1 345 18 495 Propanal 1 350 40 500-5 Formaldehyde 1 39 400 520 G1yoxa1 0.5 33 320 420-5 Pyruvaldehyde 1 70 510 450-60 Furfural 0.1 365-70 20 460-5 Glucose 1 13 395 495 Sucrose 1 460 41 515-20 Allylamine 1 355 12 505 a The same filter combination (see methods) and sensitivity (blank = 0, acrolein 0.001 pmole = 3)was used in all these measurements. * Aminco-Bowman spectrophotofluorometerused. Compounds dissolved in ethanol; all the rest are aqueous solutions. Amine concentration per ml of Tris buffer (0.05M)before the oxidation. Incubation volume 2 ml. Calf serum 0.025 ml/ml. Temp. 50 "C. Time 3 hours. Protein precipitated with TCA (14x) 0.5 ml previous to the fluorescence reaction.
VOL. 40, NO. 1 1 , SEPTEMBER 1968
1705
too
80 L.
.-mc W
c 5
60
c al
.E al
p
40
al
::
L
t
e
2c
(
5
IO
I
I
20
30
0
Time in minutes
Figure 1. Relation between fluorescence intensity and temperature
OS
5
I
1.5 3 xlo*mmolts/ml m-AMINOPHENOL CONCENTRATION
Figure 2. Fluorescence intensity in relation to the concentration of m-aminophenol in the reaction mixture Aqueous acrolein:
Aqueous solution of acrolein 0.004 pmole/ml, used for this experiment ture, while the m-aminophenol concentration was varied between 0.5 to 5 X IOe2 mmole/ml. Under these conditions, in the presence of a constant hydroxylamine concentration, a sharp increase in fluorescence yield was observed with the increase of the m-aminophenol concentration up to 1.5 x 10-2 mmole/ml. Higher concentrations were without further effect (Figure 2). Conversely, variations in the hydroxylamine concentration while maintaining constant the optimal m-aminophenol concentration, produced only slight fluctuations in the intensity of the fluorescences produced with acrolein and no changes on their excitation of fluorescence spectra. The addition of hydroxylamine to the reagent solution did produce, however, some decrease of intensity in the fluorescences obtained with a number of other compounds (Table 11), most likely through some competitive oxime formation. Furthermore, it stabilized the reagent solution for longer periods of time. Consequently, it was decided to incorporate this substance routinely into the reagent solution in spite of the fact that it was not essential for the production of fluorescence. Reference Standard Curves. A representative fluorescence calibration curve for acrolein in aqueous solution, between pmoles/ml, at a the concentrations of 0.2 and 3.4 x
8 = 0.003 pmole/ml o---O = 0.001 pmole/ml 0--0
given sensitivity (not the maximum) in the Farrand photoelectric fluorometer is depicted in Figure 3. These fluorescence curves are easily duplicated in any given set of similar conditions. Nevertheless, since some variations of intensity in the light source and/or in the fluorescences do occur with several variables (Le., time and temperature among others) two or three points on the corresponding curve for any given concentration range were redetermined each time an unknown concentration of acrolein was measured. Limits of Sensitivity of Reaction. It was possible, with the Farrand fluorometer set at maximum sensitivity, to measure the fluorescences produced with acrolein in aqueous solution pmole/ml, that is, in at concentrations as low as 5 X amounts of circa 0.002 pg/ml. With this degree of sensitivity, 1 ng/ml of acrolein gave a galvanometer deflection of about 1.5 to 2 divisions. This deflection was increased to six or seven divisions per nanogram when the readings were taken with the Aminco-Bowman spectrophotofluorometer at a high sensitivity setting (meter multiplier switch, position 0.001 ; sensitivity-control reading, 50). For crotonaldehyde, the lowest concentrations detectable pmole/ml. were of the order of 1 x Fluorescence Spectra of Resulting Compounds. The spectra of the fluorescent products from acrolein and from croton-
Table II. Effect of Hydroxylamine upon Several of the Fluorescences" Obtained with m-Aminophenol Compound Reagents (mmole/ml reaction mixture) (Aqueous sol.) m-Aminophenol, rn-Aminophenol 1.5 X 10-2 plus hydroxylamine 1.5 x 10-2 1.15 3.5 4.6 7 x 10-2 25 22 24 . 2? 23 0.001 jtmole/ml
Acrolein Acrolein 0.003 Formaldehyde 1 Acetaldehyde 1 Glyoxal 1 Furfural 1 Sucrose 1 a Farrand setting: blank 1706
68 65 34 94 85 44
62 45 19 54 12 46
68 35 17 40 57
65 31 16 35 43 39
41 = 0, acrolein 0.001 pmole/ml = 2. Each value is the media of at least two determinations.
ANALYTICAL CHEMISTRY
63 26 15 27 46 36
100
-
9,
.U
CI
en
80
c
E
80
0
a2
a
0
E L
W
60
60
W
3
8-
E
a2
W
0
c
40
In W
-z
0)
0
3
In
2
-a 0
40
0 c a2 0
Y
2c
LL
20
0
LL
250
350
450
Wavelength
650
550
- Millimicrons
I
I
I
I
I
I
1.4
1.8
2.2
2;6
30
3.4
Figure 4. Excitation and fluorescence spectra of the fluorescent products from acrolein (a) and from crotonaldehyde (c) at pH 7
ACROLEIN CONCENTRATION, Jtmolo/ml X l o "
(1 ml of the fluorescent reaction mixture plus 0.9 ml of N NaOH mixed with 3 ml of Tris Buffer 0.1M)
I
I
I
I
I
0.2 0.4 0.6 OB 1.0
Figure 3. Relation between the amount of acrolein assayed and the fluorescence produced
aldehyde at the acid pH (0.5) of the reaction mixture, did look superficially alike. However, the excitation and fluorescence maxima for the crotonaldehyde product were 5 to 10 mp below the corresponding values for the acrolein derivative (see Table I); in addition, in the 300-320 mp region only a soft bulge could be observed instead of the clear incurvation or small secondary peak normally present in the acrolein derivative excitation spectrum. These minor differences between these two fluorescent products were replaced by marked differences when the spectra were taken at a neutral pH (range 6.6-7.6) as illustrated in Figure 4. The excitation spectrum (a) in Figure 4 with its pronounced secondary peak at 330 mp, appears, so far, to be specific for the acrolein product. For example, propanal and 3-propylacrolein (2-hexenal) fluorescent derivative spectra also were similar to the spectra from the acrolein compound at the acid pH of the reaction mixture; at neutral pH however, they became different, adopting the general configuration of the spectra from the crotonaldehyde derivative [Figure 4, (c)]. The spectra of the fluorescent assay mixtures of the other compounds tested, with the exception of the compounds that generate acrolein as previously specified, differed from the spectra of acrolein either in their maxima, their configuration, or both (Table I). Consequently, the spectra from the acrolein product appeared highly specific, a finding that agreed well with the identification results. Isolation and Identification of Fluorescent Product Obtained with Acrolein as 7-Hydroxyquinoline. The fluorescent compound derived from the reaction of acrolein with m-aminophenol, was easily adsorbed in activated charcoal. From there, extraction with ethanol or butanol produced a purer and more concentrated fluorescent solution compared with the original reaction mixture. Evaporation to dryness gave a solid residue which, upon redissolving, reproduced the original fluorescences without any change in their spectral characteristics. This made possible detailed fluorescence
spectral studies with several solvents, concentrations, and different pH values. By variation of these three parameters, numerous spectra of the solid material in solution were examined and compared with the corresponding spectra of model compounds which were thought likely to represent the fluorescent principle. The results from these comparisons conclusively showed that the spectra from the purified fluorescent material of the reaction of acrolein with m-aminophenol were identical with those of 7-hydroxyquinoline in all solvents, concentrations and pH values used (Le., Figure 5 ) . As a corollary to this finding, the spectra of the acrolein reaction mixtures before undergoing any purification process, such as depicted in Figure 4, could be reproduced exactly, just by the addition of corresponding amounts of 7-hydroxyquinoline to the blanks. In subsequent steps, from the solid residua of the acrolein
r
loo L
Wavelength
-
Millimicrons
Figure 5. Excitation and fluorescence spectra at pH 14 ( N NaOH solution) obtained with the Aminco-Bowman spectrophotofluorometer (Meter multiplier switch, position 0.03; sensitivity control reading, 50) from: the purified fluorescent fraction from the acrolein reaction (- - -)and from 7-hydroxyquinoline, 0.003 @mole/ml,(-)
-
VOL. 40, NO. 1 1 , SEPTEMBER 1968
0
1707
reaction, 7-hydroxyquinoline was isolated by sublimation at atmospheric pressure. The micro-crystals from the sublimate obtained in the 190-230 "C temperature range (sublimation was performed twice ; contaminants were eliminated below these temperatures), represented 7-hydroxyquinoline. Its identity was established by the following studies: a. Comparison of the excitation and fluorescence spectra of the purified compound under study with the spectra of an authentic sample of 7-hydroxyquinoline in any given solvent or pH value (Le., Figure 5). Their excitation maxima, in mp (with secondary peaks between parentheses), were: at pH 1, 350 (245); at pH 7,400 (330,260); at pH 14, 360 (280, 250); and their fluorescence maxima 505-10,510, and 485-90 mp, respectively. b. Comparison of the UV spectra at several pH values and in different solvents. Their ultraviolet spectra curves (taken in a Cary Model 11 spectrophotometer) were characterized: in 0.01N HCI, by a sharp absorption peak at 242 mp and a secondary one at 346 mp; in 0.01NNaOH by a main peak at 243 mp and a secondary peak at 357 mp; and in 9 5 x ethanol, by an absorption maxima at 228 mp and a secondary one at 336 mp wavelength. Thus, the bathochromic shifts in acid and basic solutions relative to their absorption in neutral alcohol described for phenolic quinolines (8), was clearly observed. The above curves, in addition, corresponded accurately with the curves described in the literature as obtained with 7-hydroxyquinoline (8). c. Both compounds gave the same R, values in paper chromatograms (i.e., in isopropanol-acetic acid-water ; 60:5:35, = 0.89). d. The range of minimal concentrations that could be measured fluorometrically were practically the same for acrolein (through its fluorescent product) and 7-hydroxyquinoline. (8) G. W. Ewing and E. A. Steck, J. Am. Chem. SOC.,68, 2181
(1946).
1708
ANALYTICAL CHEMISTRY
By comparison, in the Aminco-Bowman spectrophotofluorometer, of the values of the fluorescence intensities from the reaction of minimal amounts of acrolein (1 x 10'8 to 5 x pmole/ml), with equivalent amounts of 7-hydroxyquinoline, it was concluded that around 5 5 x of the acrolein present in the highly diluted solutions was converted into 7-hydroxyquinoline under the experimental conditions described. It appears that the reaction responsible for the formation of this fluorescent compound can be included in the Doebner-Miller group of reactions for quinolines syntheses (9).
In this study, the general reaction where an m-hydroxyaniline is made to react in acid solution with an +unsaturated carbonyl compound or substances which can be converted into a,@-unsaturated compounds, then, can be represented by the following equation:
Reagents
Final product
Consequently all the compounds that can generate acrolein, such as those listed in the first section of Table I will produce 7-hydroxyquinoline as a final product. The substituted acroleins or compounds that can generate them (middle section in Table I) will most likely produce the corresponding substituted 7-hydroxyquinolines. The rest of the compounds gave lesser fluorescences that have not been further studied. RECEIVED for review May 6, 1968. Accepted June 6, 1968. Presented in part at the Federated Societies for Experimental Biology, April 1967. Investigation supported in part by Research Grant No. C-6516 from the National Cancer Institute, National Institute of Health. (9) G. M. Badger, H. P. Crocker, B. C. Ennis, J. A. Gayler, W. E. Matthews, W. G. C. Raper, E. L. Samuel, and T. M. Spotswood, Aust. J . Chem., 16, 814 (1963).
