ANALYTICAL CHEMISTRY, VOL. 50,
clohexanone, acetone, and pinacolone seemed to be apparently stable under the condition. Although the acid amide group of the a-adduct was stable a t 0 "C in the alkaline reaction mixture as stated above, alkaline hydrolysis of the amide group was observed a t higher temperature generating ammonia. +
CH3
NaOH-
+NH3
R
In all compounds tested, the final fluorescence was in inverse proportion to the amounts of ammonia liberated which was semiquantitatively determined with the Nessler reagent-filter paper. The greenish fluorescence observed with time in the alkaline reaction mixture seems to be due to a-carbinol of N M N (14) which is weakly fluorescent and almost diminished by heating in acid to liberate NMN. On the other hand, prolonged incubation or heating of the alkaline reaction mixture caused strong bluish fluorescence which cannot be quenched by acid any more. This irreversible fluorescence is considered due to side reaction of the aldehyde produced by opening of the pyridine ring of a-carbinol of N M N between N1 and C2. T h e high specificity of the present reaction is due to simultaneous requirement of two different functional groups, active methylene and adjoining carbonyl groups, for the formation of fluorophores. Therefore, indole and pyrrole, which can be detected with colorimetric reagents for active methylenes such as sodium nitroprusside (23, 24). 1,2naphthoquinone-4-sulfonic acid (25, 26) and p-dimethylaminobenzaldehyde (25), are not detectable in the present fluorescent reaction. Moreover, some other typical active methylenes such as creatinine, resorcinol, orcinol, and phloroglucinol which may produce a cyclic a-methylene carbonyl group and show positive response to the colorimetric reagents or some cyclic a-methylene carbonyls such as dimedone, camphor, and isoketopinic acid gave no fluorescence, probably because of t h e steric hindrance around the amethylene carbonyl groups. Of the expected positive compounds 1,3-diketones were nonfluorescent or weakly fluorescent in the present reaction. This may be due to the isomerization (27) of the compounds into stable enol forms. T h e application of N M N as a fluorogenic labeling reagent for carbonyl compounds in high performance liquid chromatography has been successfully performed with both
NO. 14, DECEMBER 1978
2051
pre-column and post-column derivatization methods (28). Supplementary Material Available: A table (6 pages) containing fluorescence data for various classes of carbonyl compounds after reaction with NMN followed by heating in acid will appear following these pages in the microfilm edition of this volume of the journal. Photocopies of the supplementary material from this paper or microfiche (105 X 148 mm, 24X reduction, negatives) may be obtained from Business Operations, Books and Journals Division, Americal Chemical Society, 1155 16th St., N.W., Washington, D.C. 20036. Full bibliographic citation (journal, title of article, author) and prepayment, check or money order for $5.50 for photocopy ($7.00 foreign) or $3.00 for microfiche (S4.00 foreign) are required.
LITERATURE CITED B. Camber, Nature (London), 174, 1107 (1954). P. S. Chen. Jr., Anal. Chem.. 31, 296 (1959). R. Brandt and N. D. Cheronis, Microchem. J . , 5 , 110 (1961). R. Brandt, J. C. Kouines, and N. D. Cheronis. Microchem. J . , 6, 519 (1962). R. Brandt and N. D. Cheronis, Microchin?.Acta. 3, 467 (1963). R. Brandt, J. C. Kouines, and N. D. Cheronis, J . Chromatogr., 12, 380 (1963). D. J. Pietrzyk and E. P. Chan, Anal. Chem., 42, 37 (1970). V. Graef, Z . Klin. Chem. Klin. Biochem., 8, 320 (1970). R. Chayen, R. Dvir, S. Gould, and A. Harell, Isr. J . Chem., 8, 157p (1970). R. Chayen, R. Dvir, S . Gould, and A. Harell, Anal. Blochem., 42, 283 (1971). D. N. Kramer, L. U. Tolentino, and E. B. Ha'ckley, Anal. Chem., 44, 2243 ( 1972). J. Bartos, Ann. Pharm. Fr., 27, 691 (1969) M. Pesez and J. Bartos, Ann. Pharm. F r . , 27, 161 (1969). J. W. Huff, J . Blol. Chem., 167, 151 (1947). J. W. Huff and W. A. Perlzweig, J . Biol. Chem., 167, 157 (1947). N. Levitas, J. Robinson, F. Rosen, J. W. Huff, and W. A Perlzweig, J . Biol. Chem., 167, 169 (1947). J. Robinson, N. Levitas, F. Rosen. and W. A. Perlzweig, J . Biol. Chem., 170, 653 (1947). K. J. Carpenter and E. Kodicek, Biochem. J . , 46, 421 (1950). H. B. Burch, C. A. Storvick, R. L. Bicknell, H. C. Kung, L. G. Alejo, W. A. Everhart, 0. H. Lowry, C. G. King, and 0.A. Bessey, J . Biol. Chem., 212, 897 (1955). B. R. Chrk, R. M. Halpern, and R. A. Smith, Anal. Biochem.,68, 54 (1975). M. J. Johnson, J . Biol. Chem., 137, 575 (1941). M. Ishidate and T. Sakaguchi, J . Pharm. SOC. Jpn., 70, 444 (1950). E. Legal, Jahresber. forfschr. Chem., 1648 (1883). B. Bitto, Ann. Chem., 267, 372 (1883). P. Ehrlich and C. A. Herter, Z . Physiol. Chem., 41, 329 (1904). F. Feigl and 0. Frehden, Microchemie, 16, 79 (1934). M. Ishidate and Y. Yamane. Chem. pharm. Bull. (Tokyo), 8, 1116 (1960). H. Nakamura and 2. Tamura, J , Chromatogr., in press.
RECEIVED for review July 5, 1978. Accepted September 5, 1978. Presented in part a t the 98th Annual Meeting of the Pharmaceutical Society of Japan, Okayama, April 3-5,1978. T h e authors are grateful to the Pharmacological Research Foundation, for partial support of this work.
FIuorimet ric Determination of Pyridine after Hydroxylation with the Hamilton Hydroxylation System Michelle P. Wong and Kenneth A. Connors" School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53706
Pyridine and some substituted pyridines were subjected to the Hamilton hydroxylation system, consisting of hydrogen peroxide, catechol, and ferric ion. The resulting hydroxylated pyridines were measured fluorimetrically. The method is applicable to lo-' to M aqueous solutions of several pyridine compounds; linear fluorescence concentration plots were obtained.
T h e ability of the Hamilton hydroxylation system (1-3), 0003-2700/78/0350-2051$01.00/0
consisting of catechol, ferric perchlorate, and hydrogen peroxide, to introduce a hydroxy group on an aromatic ring is potentially useful analytically. This hydroxylation system was previously used ( 4 , 5 ) as a reagent for the analysis of aqueous solutions of aromatic compounds by their conversion t o phenols, which were subsequently determined colorimetrically. The purpose of the present research was to carry out the hydroxylation step and then to measure the resulting hydroxylated product fluorimetrically, because many hydroxy compounds fluoresce strongly. Attention was restricted to @ 1978 American Chemical Society
2052
ANALYTICAL CHEMISTRY, VOL. 50, NO. 14, DECEMBER 1978
4:i
3
2
1
ib
1
; O Time
30
4'0 5 0
I
I
1
1
60
in minutes
Figure 1. Change of fluorescence intensity with time for hydroxylation M pyridine, of pyridine at 80 " C . Initial conditions were: 1.98 X 6.0 X M ferric perchlorate, 1.2 X M catechol, pH 4.0, [H,O,]/[pyridine] ratio: 0, 35.4; C3,17.7; and 0 , 3.54. Wavelengths nm were X,,300 nm, X,,350
3-
pyridine compounds, which could not be determined by the earlier method, and which do not fluoresce strongly, whereas hydroxypyridines do.
EXPERIMENTAL Materials. All inorganic chemicals were analytical reagent grade, and were used without further purification. Ferric perchlorate hexahydrate (Fe(C10&-6H20)was obtained from G. F. Smith Co. The following chemicals, listed according to source, were purified before use. 2-Picoline (Aldrich) was distilled, bp 127-129 "C (lit. ( 6 ) ,128.8 "C); quinoline (Aldrich) was double distilled a t reduced pressure; 2,6-pyridinedicarboxylic acid (Aldrich) was recrystallized from water, mp 252 "C (lit. (71, 252 "C); 4-dimethylaminopyridine (Aldrich) was recrystallized from Skellysolve A, mp 113-114 "C (lit. (7),114 "C); catechol (Eastman Organic Chemicals) was recrystallized twice from toluene, mp 103-104 "C (lit. (81,104 "C). Reagent grade pyridine was obtained from Merck Chemical Co., and Certified A.C.S. pyridine was obtained from Fisher Scientific Co. Acetic acid (Mallinckrodt) was analytical grade. Sodium acetate (J. T. Baker Chemical Co.) meets A.C.S. specifications. All other organic chemicals were obtained from Aldrich Chemical Co. and were used directly. Apparatus. Fluorescence measurements were made with a Perkin-Elmer MPF-4 Fluorescence Spectrophotometer in the energy mode with the excitation and emission slits set at 10 nm. The instrument was fitted with thermostated cell compartments that maintained temperature at 25 f 0.1 "C. pH measurements were made at 25.0 "C with an Orion Model 801 pH meter. Analytical Procedure. To a 25-mL volumetric flask were added 1.0 mL of 3 X M aqueous catechol, 1.0 mL of 1.5 X M ferric perchlorate, and 20.0 mL of a solution of the heterocyclic compound such that its concentration in the diluted solution would be in the range to M. The solution was diluted to the mark with pH 4.0 acetate buffer (0.005 M in total buffer concentration, ionic strength 0.1 M adjusted with potassium chloride). The pH of this reaction solution should be in the range of 3.9 to 4.1. This flask was placed in a 80 "C water bath and reaction was initiated by adding 0.1 mL of 3% hydrogen peroxide solution. After 10 min at 80 "C, a 20.0-mL aliquot was pipetted into 5.0 mL of pH 12.8 borate buffer (0.4 M sodium borate + 0.2 h'I sodium hydroxide). This solution was then brought to 25 "C before the fluorescence was measured. Fluorescence intensity was read at the optimum excitation and emission wavelengths. The fluorescence of a reagent blank carried through the same procedure was also measured. A standard curve was prepared by subjecting known concentrations of the same heterocyclic compound to the procedure. Optimization Studies. During the development of the proposed method, the reaction pH and the concentrations of hydrogen peroxide, catechol, and ferric ion were varied in much
2I-
"0
2
4
6
8
IO
12
Reaction pH Figure 3. Dependence of fluorescence on reaction pH for treatment of pyridine with the Hamilton system. Initial reactant concentrations were the same as those described in Figure 1. Reaction was carried out in various pH media for 10 minutes at 80 " C . (3, Fluorescence of products measured after they were pipetted into a borate buffer of pH 12.8; 0, fluorescence of products without bringing final pH to 12.8.
the same manner as described by Albert (9). Pyridine was used as the sample in the optimization studies.
RESULTS Dependence of Yield on Peroxide/Substrate Ratio. Figures 1 and 2 show that the fluorescence intensity is maximized after 10 minutes reaction time with a n initial peroxide-to-pyridine ratio of 25 or higher. Stable fluorescence intensities were observed, as shown from the time course studies (Figure 1). I t is therefore unnecessary to add a "stabilizer", as in the earlier method for aromatic compounds. (4,5 ) . Dependence of Yield on pH. Hamilton (2, 3) indicated that the rate of loss of peroxide for the hydroxylation of anisole was independent of the p H from 3.5 to 4.2. Figure 3 shows that fluorescence was highest when the reaction was carried out at p H 4.0 and the hydroxypyridine fluoresces strongly in the anion form. Dependence of Fluorescence of Products on pH. Two hundred milliliters of 1.98 X M pyridine was subjected to the present analytical procedure. After heating the mixture a t 80 "C for 10 min, 20.0 mL aliquots were each pipetted into various buffers. T h e reagent mixture with no pyridine was subjected to the same conditions. Fluorescence intensities of the products and reagent blanks a t various pHs are shown in Figure 4. Analytical Results, Using the conditions determined from optimization studies, namely 5 X M H202,1.2 X M catechol, 6.0 X 10-5M Fe(III), in p H 4.0 buffer, reaction time 10 min a t 80 "C, 10-j to M solutions of pyridine and pyridine derivatives were treated. Table I lists the simple
ANALYTICAL CHEMISTRY,
VOL.50, NO. 14, DECEMBER 1978
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Table 111. Fluorescence Intensities of 4 x M Hydroxypyridine Isomers in pH 12.8 Borate Buffer hydroxypyridine isomers F
0-
mPa
fluorescence intensitiesa 57.4 90.5 8.1
The fluorescence intensities were corrected for buffer
blanks, sensitivity setting o n Perkin-Elmer MPF-4 fluorescence spectrophotometer was 1OX. Fluorescence measurements were all made at ,A, 300 rim and h e , 350 nm. PH
Figure 4. Dependence of fluorescence of products on pH for treatment
of pyridine with the analytical reagent. Products were brought to various pH after subjecting pyridine to the reagent for 10 min at 80 O C . 0, Fluorescence of products; 0 , fluorescence of reagents Table I. Heterocyclic Compounds Determined by the Hamilton Hydroxylation Procedure a t 80 "C analytical wavelengths, concn nm levels detersample compound hex hem mined, M pyridine 300 350 10-6-10-4 2-picoline 300 350 10-6-10-4 4-dimethylaminopyridine 340 410 10-5-10-4 2,6-pyridine-dicarboxylic acid 290 37 5 10-5-10-4 Table 11. Increase in Analytical Sensitivity for Pyridine Compounds Subjected to Hydroxylation concn of sample compound sample, M gain pyridine 5X 160 2-picoline 8 x 10.' 50 4-dimethylaminopyridine 2 x io-4 30 2,6-pyridine-dicarboxylic acid 7 x 10.' 20 heterocyclic compounds that have been successfully treated by t h e Hamilton reagent. Linear working curves of fluorescence vs. concentration were observed for the hydroxylated compounds in the concentration levels indicated. Fluorescence intensities were higher after hydroxylation for these compounds. T h e increases in analytical sensitivity for these compounds are listed in Table 11. Estimation of Product Yield and Distribution. Solutions of 4 x M o-, m-, and p- hydroxypyridines were prepared. Their final pHs were adjusted to 12.8 with borate buffer. T h e fluorescence intensities of these solutions, a t the wavelengths found optimal for pyridine analysis and corrected for t h e buffer blank, are recorded in Table 111. The hydroxylation procedure described in the analytical procedure was applied to 4 X M pyridine, and the fluorescence of t h e product corrected for the reagent blank was measured, giving a fluorescence intensity of 30.8 units with the same sensitivity settings (Aex 300 nm, A,, 350 nm, and sensitivity
lox).
It was earlier (5) reported that pyridine does not produce rn-hydroxypyridine in significant yield on treatment with the hydroxylating agent. Since the present analytical reagents and reaction conditions were identical with those used earlier (5), the products formed should also be the same as theirs. Figure 4 shows t h a t the fluorescence of products formed on hydroxylation increases with pH. Moreover, the fluorescence intensities remained t h e same from p H 12.5 to 13.0. Fluorescence spectra of individual hydroxypyridine isomers indicate that the anion forms have higher quantum yields than the protonated forms. Since the pK, values of 0 - and p-
hydroxypyridines are 11.6 and 11.1respectively, while pK, of m-hydroxypyridine is 8.7 ( I O ) , it is expected t h a t the fluorescence of products would not change significantly from p H 10 to 11 if m-hydroxypyridine is the major product. An increase in fluorescence is observed from p H 10 to 11 as shown in Figure 4. These results therefore support the conclusion that m-hydroxypyridine is not formed significantly.
DISCUSSION The Hydroxylation System. Figure 2 shows t h e extent of hydroxylation of pyridine with the Hamilton reagent as a function of t h e initial ratio of peroxide t o pyridine. Fluorescence was highest a t a [H202]/[pyridine]ratio of 25 or higher. It is not obvious why such a high ratio is necessary. Figure 3 demonstrates that an optimum p H for the hydroxylation is 4.0. This is identical with what was found earlier ( 5 ) . T h e fluorescence of the hydroxypyridine products was much enhanced when subjected to the borate buffer of p H 12.8 as shown in Figure 4. It appeared that t h e anion form of the hydroxypyridines has a high quantum yield. Independent experiments showed that the anion forms of the 0 - , m-, and p-hydroxypyridines have higher quantum yields than t h e unionized forms. I t was reported ( 5 ) that pyridine gives no color in the hydroxylation/4-aminoantipyrine colorimetric method. Separate experiments showed that 0- and p-hydroxypyridines give no color on treatment with 4-aminoantipyrine whereas m-hydroxypyridine does give a color. These results suggest that pyridine does not produce m-hydroxypyridine in significant yield on treatment with the hydroxylating agent, although the ortho and para isomers may be produced. Results from the present work show that the fluorescence of pyridine is enhanced after hydroxblation. Ortho-, meta-, and para-hydroxypyridines are more fluorescent than pyridine, as shown by the fluorescence spectra of the individual hydroxypyridine isomers. Moreover, more enhancement was observed when the hydroxypyridine products were subjected to high pH. The individual hydroxypyridine isomers also have higher quantum yields compared to their nonionic forms. The fluorescence of products as a function of p H suggested that m-hydroxypyridine is not formed predominantly. T h e fluorescence spectrum of pyridine after hydroxylation is similar to those of 0 - and rn-hydroxypyridines, but not phydroxypyridine. Both the ortho and meta isomers have an excitation maximum of 300 n m and an emission maximum of 350 nm, while the para isomer does not show any distinct excitation or emission maxima. Since the analytical reagents and reaction conditions were the same as those used earlier (5),the products formed should also be identical. Pyridine does not produce m-hydroxypyridine in significant yield. Based on these results, it is therefore highly probable that the reaction with the reagents produces mainly o-hydroxypyridine, Assuming that the product formed on hydroxylation of pyridine is o-hydroxypyridine, the percentage yield is 53.7. To test whether o-hydroxypyridine was the only product formed, 2,6-disubstituted pyridines were subjected to the Hamilton reagent. Fluorescence enhancement was obtained
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 14, DECEMBER 1978
when 2,6-pyridine-dicarboxylicacid was subjected to the reagent conditions. T h u s it appears that some p-hydroxypyridine could be formed. If so, it should nct be possible to hydroxylate a 2,4,6-trisubstituted pyridine. The fluorescence spectrum of 2,4,6-trimethylpyridine remained the same after it was treated with the reagents. Hence, if 0 - and p - isomers are the products, the yield would be 54% or greater. Analysis by Hydroxylation. T h e analytical method is applicable to several pyridine compounds tested in the range 104-10-4 M. Table I1 shows the gain in analytical sensitivity after hydroxylation of the various pyridine compounds. Gain is defined by the following expression: F(sample after hydroxylation)- F(reagent) F(samp1e
in
water) - Ftwater)
where F(sample after h&,.&tlon) = fluorescence intensity of sample after hydroxylation, F(reagent) = fluorescence intensity of reagent blank treated under the same procedure, F(jample In water) fluorescence intensity of aqueous solution of sample, and F(uaterj = fluorescence intensity of water. There is no p H dependence on F(sample ,=water) for the samples listed in Table 11. Calibration plots were linear.
Some heterocyclic compounds do not yield an analytical gain when subjected to t h e hydroxylation method. T h u s quinoline fluoresces strongly in acidic medium, whereas after hydroxylation a quinoline solution fluoresces (but less intensely) in acidic and alkaline media. T h e effect of hydroxylation is, in this case, to decrease analytical sensitivity, though the altered p H dependence of the fluorescence may prove to be analytically useful.
LITERATURE CITED (1) G. A. Hamilton and J. P. Friedman, J. Am. Cbem. Soc., 85, 1008 (1963). (2) G. A. Hamilton, J. P. Friedman, and P. M. Campbell, J . Am. Cbem. SOC., 88, 5266 (1966). (3) G. A. Hamilton, J. W. Hanifin, and J. P. Friedman, J . A m . Cbem. Soc., 88, 5269 (1966). (4) K . A. Connors and K . S. Albert, Anal. Cbem., 44, 879 (1972). (5) K. S. Albert and K. A. Connors, J . Pharm. Sci., 62, 625 (1973). (6) C. J. Eguchi, Bull. Cbem. SOC.Jpn., 2, 180 (1927). (7) R. C. Weast, "Handbook of Chemistry and Physics", 53rd ed., Chemical Rubber Co., Cleveland, Ohio, 1972. (8) H. D. Dakin, Org. Syn., 1, 143 (1932). (9) K. S. Albert, P h D dissertation, University of Wisconsin, Madison, Wis., 1972 (10) A. Albert and A. Hampton, J , Chem. Soc., 507 (1954)
RECEIVED for review May 30, 1978. Accepted September 1, 1978.
Singlet-Triplet Energy Difference as a Parameter of Selectivity in Synchronous Phosphorimetry T. Vo-Dinh" and R. B. Gammage Health and Safety Research Division, Oak Ridge National Laborafory, Oak Ridge, Tennessee 37830
The direct use of the singlet-triplet energy difference as a new factor of selectivity in phosphorimetry is suggested. The methodology, which is based on the synchronous excitation technique, exploits the specificity of energy gaps AsT between So) and the the phosphorescence emission band (T, absorption bands (S, So or S, So). This approach can greatly improve the selectivity in phosphorescence analysis. Some aspects of the specificity of the parameter AA in the characterization of polynuclear aromatic (PNA) compounds are discussed. The differentiation of isomeric PNAs is illustrated and the qualitative analysis of a Synthoil sample is given.
-
+
+
T h e performance of a n analytical method in multicomponent analysis is often characterized by the variety of selectivity factors it offers. Regarding this aspect, luminescence spectrometry offers the choice of selecting independently two wavelength variables (excitation and emission). The specificity of luminescence spectrometry can be enhanced by using special techniques to exploit these spectral variables. A modulation technique based upon a slight difference in spectral bands for selectively obtaining excitation and emission spectra of a specific component in a mixture of two fluorophores has been reported ( I ) . Other features (2, 3) of the spectral structure have also been amply exploited by using quasi-linear spectroscopy a t low temperatures, especially with Shpolskii solvents ( 3 ) and other matrices ( 4 ) . Alternatively, time-resolved and phase-resolved spectroscopies (5,6),based on the differences in the lifetime of the excited states have 0003-2700/78/0350-2054$01.00/0
opened u p new analytical possibilities. We first report here the exploitation of a physical parameter of the excited states, namely the singlet-triplet energy difference (AsT), as a direct factor of selectivity in phosphorimetric analysis. Although AST is a well-known physical concept, to the authors' knowledge it has never been exploited explicitly as a factor of selectivity. In the past, t h e energy levels of the excited singlet and triplet states of organic molecules have been used in conventional spectroscopy to characterize them, but the difference in energy between t h e excited singlets Sl(or S,) and the lowest triplet TI (from which the compound phosphoresces) has not yet been used experimentally to identify compounds in multicomponent analysis. T h e use of this singlet-triplet energy gap employs the so-called synchronous luminescence technique where the excitation and emission wavelengths are scanned simultaneously (7). In an earlier publication, a general methodology for this technique in fluorimetry was reported (8); it was demonstrated that this synchronous technique is not limited merely to providing finger-print signatures of complex samples such as oil spills ( 9 ) ,but it also can provide more specific information about t h e molecular size and type of the components. A similar approach applied to phosphorescence analysis is reported in this study. Although the experimental procedures for synchronous scanning in phosphorimetry are the same as those described previously in fluorescence analysis ( 8 ) ,the photophysical principles involved in the two types of applications are fundamentally different. T h e procedure t o "sort out" selectively a specific emission signal of one component in a mixture is described. An analysis of several C 1978 American Chemical Society
