Analytica Chimica Acta 467 (2002) 105114

4-Aminoantipyrine spectrophotometric method of phenol analysis Study of the reaction products via liquid chromatography with diode-array and mass spectrometric detection
Y. Fiamegos, C. Stalikas, G. Pilidis
Department of Chemistry, University of Ioannina, University Campus, 45110 Ioannina, Greece Received 19 October 2001; accepted 29 January 2002

Abstract The synthesis of new pyrazolone molecules and their application as chromogenic agents in phenol analysis by the 4-aminoantipyrine method has been described by our group in previous published papers. A fully detailed mechanism is given therein based on experimental and theoretical data. In this paper, the study of the reaction products and by-products via liquid chromatography with diode array and mass spectrometric detection is presented, sorting out the spectrophotometric determination of phenols. 2002 Published by Elsevier Science B.V.
Keywords: 4-Aminopyrazolones derivatives; Phenol analysis; Diode-array detection; Mass spectrometric detection

1. Introduction Liquid chromatographic techniques are preferable for the determination of organic molecules because they cover a wide range of polarities and permit the identication of different species. Liquid chromatography with diode-array detection (LC-DAD), has successfully been used to monitor pesticides of various natures such as organophosphorus, carbamated phenoxyacids and phenolic compounds [1]. The main advantages of LC-DAD are the ease of use and the rendering of some structural information that can be used to identify mixtures of organic compounds. However, certain limitations arise for compounds that have poor chromophore and the inability to identify unknown compounds in real samples. These difculties are overcome by using LC with mass
Corresponding author. Fax: +30-65198362. E-mail address: gpilidis@cc.uoi.gr (G. Pilidis).

spectrometric detection (LC-MS). Coupling of LC to MS through a particle beam (PB) gives high detection limits and poor reproducibility. Moreover, the advent of atmospheric pressure chemical ionization (APCI) LCMS interfaces has overcome the disadvantages of GCMS and other LCMS interfacing devices, because they can provide strong structural information similar to those obtained by the chemical ionization. Phenol analysis by liquid and gas chromatography is sensitive and selective. The well known 4-aminoantipyrine spectrophotometric method [2] developed in 1943 by Emerson [3], is still in common use mainly due to special features like speed, cost-effectiveness and absence of laborious steps. Since Emersons pioneering work on the color reaction of 4-aminoantipyrine, the study of the products formed was done by classical physicochemical methods or by the use of thin layer chromatography (TLC) [47]. In previous papers published by our group,

0003-2670/02/$ see front matter 2002 Published by Elsevier Science B.V. PII: S 0 0 0 3 - 2 6 7 0 ( 0 2 ) 0 0 0 7 2 - 7

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the sensitivity and selectivity of the spectrophotometric method were studied [8,9]. Six 4-aminopyrazolone compounds were synthesized [10] and their complex products with phenol were monitored by their color intensity (absorbance). In the present work, the quinonoid products formed by the reaction of the six newly synthesized compounds are studied in order to identify them while colored by-products are taken into consideration to assess their interference in the spectrophotometric analysis. The IUPAC names of these pyrazolonic compounds along with their abbreviations are given below: 4-AAP (1): 4-amino-1,5-dimethyl-2-phenyl-1,2dihydro-3H-pyrazol-3-one; DAA (2): 4-amino-2-(4-aminophenyl)-1,5dimethyl-1,2-dihydro-3H-pyrazol-3-one; 4-AEP (3): 4-amino-1-ethyl-5-methyl-2-phenyl1,2-dihydro-3H-pyrazol-3-one; DAP (4): 4-amino-5-(4-aminophenyl)-1-methyl-2phenyl-1,2-dihydro-3H-pyrazol-3-one; DADP (5): 4-amino-2-(4-aminophenyl)-1-methyl5-phenyl-1,2-dihydro-3H-pyrazol-3-one; 1,2-D (6): 4-amino-5-methyl-1,2-diphenyl-1,2dihydro-3H-pyrazol-3-one; 2,5-D (7): 4-amino-1-methyl-2,5-diphenyl-1,2dihydro-3H-pyrazol-3-one;

2. Experimental 2.1. Procedural details on the reaction of 4-aminopyrazolones with phenol and o-cresol Each of the seven 4-aminopyrazolone molecules with 4-aminoantipyrine included (Fig. 1), are reacted with phenol and o-cresol which are taken as model compounds, in aqueous solutions. Specically, in 75 ml of aqueous solution (buffered to pH = 9.0 with the use of 5 ml of 0.2 M) hydrogen/dihydrogen phosphate solution, 1.0 mmol of phenol and 1.5 mmol of the pyrazolone, 10 mmol of potassium hexacyano ferrate (III) is added. The solution is stirred vigorously for 30 min, diluted to 200 ml with water and extracted with 3 20 ml of CHCl3 . The organic layer is dried and the solvent is removed under a gentle stream of argon. The purple-red oily residue is sealed under argon and stored at 4 C until analyzed. 2.2. Equipment and chromatographic method The condensed products is analyzed by LCDAD, thermal beam electron impact/MS (TB-EI/MS) and APCI-MS. Blank solutions are run simultaneously and the isolated mixtures analyzed by LC with all three

Fig. 1. Generic reaction scheme for the six pyrazolonic compounds along with 4-AAP which were synthesized, characterized and used as chromophores in the spectrophotometric method for phenols (R3 = H) analysis. Abbreviation explanations are given in the text.

Y. Fiamegos et al. / Analytica Chimica Acta 467 (2002) 105114 Table 1 The gradient program of the solvent used for the analysis Time (min) 0.00 15.00 20.00 28.00 30.00 Methanol (%) 80 40 10 80 80 Water (%) 20 60 90 20 20

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detectors. For the main products detected with LC-APCI ()-MS the respective MSMS spectra are also studied. The analyses were performed with a thermo separation gradient pump LC system, with a Supelcosil LC-18-DB, 2.1 mm 25 mm, 3 m (Supelco) at 25 C. The gradient program used is shown in Table 1. The ionization was done at an

APCI temperature of 450 C with the corona discharge at 5.5 kV. The MS detection was made by a Finnigan MAT LCQ ion trap mass spectrometer. The UVVIS LC-DAD (Waters 996) was connected on-line with the TB-EI/MS detector (Waters Integrity System, Milford, MA). Ionization was at 70 eV, the source was at 230 C and the temperature of the TB interface was set at 88 C. The tandem mass spectra were acquired using the infusion technique into the APCI device at 30 l min1 . The 4-aminoantipyrine-phenol quinone-imide product was isolated as described elsewhere [11]. The respective reactions with the other pyrazolonic compounds were repeated and the reddish remainder obtained after the evaporation of the extraction solvent was subjected to column chromatography in order to isolate the corresponding quinonoids (Fig. 2(B),

Table 2 Product ions derived from single and multiple mass spectrometric analysis of 4-aminopyrazolonesa 4-Aminopyrazolone 4-AAP (MW 203) MS (m/z) 203 30) 202 ([MH] , 100) (M , MSMS (m/z) 203 173 109 92 (60) (25) (95) (100) EI (m/z) 203 (M+ , 100) 119 (15) 93 (40) 218 (M+ , 90) 134 (30) 119 (10) 217 216 203 119 280 160 133 120 280 256 160 120 265 172 145 118 265 146 117 104 (M+ , 100) (100) (10) (10) (M+ , 60) (30) (70) (100) (M+ , 80) (60) (90) (100) (M+ , 100) (70) (90) (85) (M+ , 100) (95) (90) (85) 84 (95) 77 (85) 57 (95) 108 (30) 84 (85) 57 (100) 98 (60) 71 (90) 56 (85) 92 77 65 51 (50) (25) (30) (20)

DAA (MW 218)

218 (M , 10) 217 ([MH] , 100) 216 (70) 217 (M , 40) 216 ([MH] , 100)

216 (85) 188 (100) 133 (40) 216 (100) 123 (75) 93 (50) 278 255 185 99 (100) (40) (5) (20)

4-AEP (MW 217)

DAP (MW 280)

280 (M , 15) 279 ([MH] , 100)

DADP (MW 280)

280 (M , 10) 279 ([MH] , 100)

279 (85) 265 (100) 171 (40) 265 (100) 237 (70) 146 (20) 265 (40) 250 (100) 171 (75)

93 (65) 77 (45) 51 (30) 104 93 77 51 91 77 63 51 (75) (35) (85) (90) (50) (75) (20) (80)

1,2-D (MW 265)

265 (M , 30) 264 ([MH] , 100)

2,5-D (MW 265)

265 (M , 30) 264 ([MH] , 100)

Relative ion intensity, %, is reported in parentheses.

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Fig. 2. The basic fragmentation between the pyrazolonic compound (A) (4-AAP) and its quinonoid analogue (B), with o-cresol.

IUPAC name: 1,5-dimethyl-4-[(4-oxocyclohexa-2,5dien-1-ylidene)amino]-2-phenyl-1,2-dihydro-3H-pyrazol-3-one). The elution system used was ethyl acetate/petroleum ether (1/44/1). These six quinonoids were isolated and characterized via their spectro-

metric data (IR, 1 H NMR, 12 C NMR, MS) and their purity was conrmed by the use of LC-APCI-MS as >98%. These puried products were utilized as standard compounds for the quantication of the products formed during spectrophotometric phenol analysis.

Table 3 Product ions derived from single and multiple mass spectrometric analysis of colored quinone-imide compounds formed during phenol analysisa Quinone-imide 4-AAP-phenol (MW 293) MS (m/z) 293 (M , 65) 292 ([MH] , 100) 308 (M , 75) 307 ([MH] , 100) 307 (M , 30) 306 ([MH] , 100) 371 (50) 370 (M , 100) 369 ([MH] , 45) 371 (45) 370 (M , 10) 369 ([MH] , 100) 356 (20) 355 (M , 100) 354 ([MH] , 25) 356 (20) 355 (M , 20) MSMS (m/z) 293 (100) 278 (60) 250 (90) 308 (70) 292 (25) 279 (20) 306 (100) 276 (50) 264 (25) 370 (100) 343 (30) 279 (25) 370 355 342 311 (80) (60) (40) (30) 236 (75) 175 (50) 266 (20) 173 (100) 236 (20) 186 (15) 160 (10) 250 (30) 185 (40) 117 (25) 262 235 201 134 237 (100) 171 (45) (60) (100) (50) (25)

DAA-phenol (MW 308)

4-AEP-phenol (MW 307)

DAP-phenol (MW 370)

DADP-phenol (MW 370)

1,2-D-phenol (MW 355)

356 (100) 314 (10) 237 (40) 356 (85) 340 (60) 236 (60)

2,5-D-phenol (MW 355)

Relative ion intensity, %, is reported in parentheses.

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3. Results and discussion The UVVIS (200800 nm), APCI-MS/(MSMS), and TB-EI/MS spectra of the seven studied 4-aminopyrazolone molecules were evaluated by dedicated

software. None of these compounds interferes with the quinone-imide absorbance at 420510 nm. The MS data of the pyrazolones is shown in Table 2. Most of them in the EI spectra shows the existence of the fragment ion m/z: 119 or (MW 119)+ due to the possible

Table 4 Analytical features for the spectrophotometric method of phenol determination with the use of different 4-aminopyrazolones Phenol reacted with 4-AAP DAA 4-AEP DAP DADP 1,2-D 2,5-D Application in spiked samples. a Mean S.D.; n = 5. Calibration equation y y y y y y y = 37080 x 10069 = 32430 x + 10326 = 66740 x 36428 = 20730 x + 3604 = 31860 x 9670 = 51880 x 25226 = 160830 x + 93167 r2 (n = 6) 0.999 0.998 0.999 0.998 0.999 0.999 0.998 Phenol analysis (50 mg l )a 50.0 49.7 50.8 49.9 49.8 50.4 49.5 0.4 0.4 0.6 1.1 0.1 1.0 0.7

Fig. 3. LCMS chromatogram of reaction mixture of 1,2-D with 50 g l1 phenol and the respective mass spectra of the peaks eluting in SIC mode. The MS were obtained in the APCI mode.

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bond cleavage of the pyrazolonic ring in position NN and C3 C4 = (Fig. 2). The reaction mixtures of pyrazolones with phenol were analyzed by LC-APCI-MS and MSMS. In all cases, the target product is formed in small quantities with by-products being in excess [1113]. The MS and MSMS ion fragments of the derivatized phenols are summarized in Table 3. Most frequently, the formed molecular ions in this mode (APCI-negative mode, APCI) are M and [MH] . Surprisingly, in the

case of quinone-imides DAP-phenol, DADP-phenol, 1,2-D-phenol and 2,5-D-phenol, the ion [M+H] ion is observed too. Calibration graphs were drawn by the external standard calibration method in the concentration range 5500 g l1 . Regression lines were calculated by averaging the data used for the calibration graphs each day. As shown in Table 4, the linearity is good for all quinonoid products with correlation coefcients (r2 ) >0.998. It is noteworthy that the measured values

Table 5 Product ions derived from multiple mass spectrometric and EI analysis of the possible pyrazolonic dimers formed during phenol analysisa Compounds 4-AAP-dimer (MW 2 203) MS (m/z) 402 (30) 401 (100) MSMS (m/z) 401 370 308 283 431 388 321 298 430 429 384 336 310 559 395 276 255 542 541 422 409 528 510 499 435 344 225 530 529 472 410 395 291 (100) (80) (35) (50) (90) (25) (100) (10) (100) (80) (40) (55) (30) (50) (100) (30) (30) (50) (100) (50) (15) (30) (100) (25) (50) (40) (20) (15) (85) (10) (100) (35) (10) EI (m/z) 402 228 137 119 432 248 187 134 (60) (80) (15) (10) (5) (15) (15) (60) 104 (50) 83 (75) 56 (100) 108 79 67 56 (45) (30) (55) (100)

DAA-dimer (MW 2 218)

432 (20) 431 (100)

4-AEP-dimer (MW 2 217)

430 (20) 429 (100)

DAP-dimer (MW 2 280)

560 (30) 559 (100) 558 (75) 542 (45) 541 (100) 540 (90) 529 (30) 528 (100)

DADP-dimer (MW 2 280)

1,2-D-dimer (MW 2 265)

2,5-D-dimer (MW 2 265)

530 (15) 529 (100)

a b

Relative ion intensity, %, is reported in parentheses. , information was not obtainable.

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of the phenol contents in spiked water samples is in satisfactory agreement with the added concentration (50 g l1 ). In all mixtures, two main products were observed in comparatively high quantities (Fig. 3), one of them being the dimer of the employed 4-aminopyrazolone (Table 5). By analogy, the reaction mixtures of pyrazolones with o-cresol gave one quinone-imide (Table 6) with dimers in large quantities. In all cases, the only colored products identied by LC-DAD analysis were the respective quinone-imide products absorbing as expected between 420 and 510 nm. Relevant molecular spectra are provided in Fig. 4. The second amino group (p-aminophenyl) of diaminopyrazolones DAA, DAP and DADP seems not to react with phenol since no diquinone-imides or multiple quinonoid compounds were detected. The reaction of 4-AEP with o-cresol gave a second possible quinone-imide, which does not absorb between 400 and 500 nm.

The analysis of the blank reaction mixtures conrmed the formation of dimers in all cases. None of them absorbs above 430 nm. This formation seems to be independent of the chromogenic reaction with phenol and probably is much slower because phenol analysis gives reproducible results. The structure of these dimeric products may be related to azo- and diazo compounds but certainly are not pertinent to antipyrine red [14]. 3.1. Investigation of the p-aminophenyl group behavior in the analysis The behavior of the second amino group (p-aminophenyl) of diamino pyrazolones DAA, DAP and DADP was studied by synthesizing the monoamino products (Fig. 5) and repeating the reaction with phenol and o-cresol (in the absence of an amino group in position 4). The molecules of 2-(4-aminophenyl)-1,5-dimethyl1,2-dihydro-3H-pyrazol-3-one (8),5-(4-aminophenyl)-

Fig. 4. UVVIS spectra of the colored quinone-imides formed by the reaction of 2,5-D, 4-AAP, DAA and 1,2-D with phenol.

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Table 6 Product ions derived from EI mass spectrometric analysis of the colored quinone-imide compounds of o-cresola Compounds 4-AAP-cresol (MW 307) EI (m/z) 307 (M+ , 80) 294 (10) 189 (15) 324 309 189 134 321 308 189 134 (10) (10) (10) (15) (25) (15) (15) (10) 134 (10) 77 (30) 56 (100) 108 77 56 91 70 56 (20) (15) (100) (10) (100) (25)

DAA-cresol (MW 322)

4-AEP-cresol (MW 321)

DAP-cresol (MW 384)

386 (30) 266 (20) 133 (100) 384 280 133 120 371 354 265 210 172 (50) (100) (90) (30) (5) (5) (20) (10) (20)

119 (30) 93 (60) 77 (50) 56 (30) 145 118 104 77 51 (25) (100) (20) (70) (60)

1-methyl-2-phenyl-1,2-dihydro-3H-pyrazol-3-one (10) and 2-(4-aminophenyl)-1-methyl-5-phenyl-1,2-dihydro-3H-pyrazol-3-one (11), in their reactions with phenol and o-cresol gave possible quinone-imide products. Interestingly, only in the case of compound (11) does the product with o-cresol absorbs at 450 nm. The fact that the respective diaminopyrazolones DAA, DAP and DADP did not result in diquinoid or multiple quinonoid products leads to the conclusion that the occurrence of the 4-amino group in the pyrazolonic ring renders the second amino group (p-aminophenyl) unreactive. In contrast, aminopyrazolone (9) did not react at all with phenol and o-cresol. It was not possible to recover the parent molecule from the reaction mixture, which seems to have been transformed fully to a dimer. [LCMS (APCI) m/z: 501 (10), 500 (65), 499 (100)]. 3.2. The behavior of para-substituted phenols Emerson has stated that certain p-substituted halo-, hydroxy- and methoxy-phenols during their reaction with 4-AAP expel the p-substituent or produce the meta or ortho transfer products. In order to clarify this phenomenon, phenol, 4-chlorophenol, 4-hydroxyphenol and 4-methoxyphenol independently are brought together with 4-AAP. The reaction mixtures were isolated and analyzed as above. The chromatograms of these four mixtures are almost identical and the only quinone-imide identied is 4-aminoantipyrine-phenol.

DADP-cresol (MW 384)

1,2-D-cresol (MW 369)

2,5-D-cresol (MW 369)

371 (10) 265 (10) 146 (10)

118 (100) 104 (40) 77 (45)

Relative ion intensity, %, is reported in parentheses.

Fig. 5. The four monoamino compounds which were synthesized and tested for their ability to produce quinone-imides.

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Fig. 6. The reaction of 4-AAP with 50 g l1 phenol, p-chlorophenol, 4-hydroxybenzoic acid and p-methoxyphenol (Figure 1, R3 = H, Cl, OH, MeO, respectively).

Fig. 7. Substitution of the 4-chloro group of the phenolic ring in the determination of 2,4- and 3,4-dichlorophenol with 4-AAP.

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Table 7 The 4-AAP-phenol compound formed during the determination of 50 g l1 of phenol, p-chlorophenol, 4-hydroxybenzoic acid and p-methoxyphenol Phenolic compounds (50 g l1 ) Phenol 4-Chlorophenol 4-Hydroxyphenol 4-Methoxyphenol
a

4-AAP-phenol quinone-imidea 49.93 47.43 46.89 39.03 0.57 0.44 0.89 1.04

Mean S.D.; n = 3.

From this fact, we can infer that p-substituents are actually expelled (Fig. 6). The quantication of these quinone-imides (Table 7) showed that when phenol is p-substituted, the reaction is not quantitative. The recovery of the phenolic compound is poorer and hence the method lacks selectivity and sensitivity. The same conclusion is drawn by the analysis of the reaction mixtures of 2,4-dichlorophenol and 3,4-dichlorophenol with 4-AAP. In both cases, the only quinone-imides observed were the respective 2-chloro- and 3-chloroquinonoids (Fig. 7). Importantly enough, similar behavior was observed when 4-AAP is replaced with the synthesized pyrazolone compounds.

position of the pyrazolone ring seems to react more readily with phenolic compound without allowing the formation of other quinonoid by-products. Theoretically, the 4-amino group is more reactive due to its participation in a conjugated system present in the pyrazolone ring, which stabilizes intermediate cationic forms or radicals. All 4-aminopyrazolones form dimers as by-products. None of them absorbs in the range of 420 500 nm which is critical for the spectrophotometric analysis. The numerous by-products of the reactions are colorless and therefore do not interfere in the method. The p-substituted phenols form substitution products during their reaction with 4-aminopyrazolones. References
[1] M.W.F. Nielen, U.A.T. Brinkman, R.W. Frei, Anal. Chem. 57 (1985) 806. [2] Standard Methods for the Examination of Water and Waste Water, 17th Edition, American Public Health Association, New York, 1989, pp. 551. [3] E. Emerson, J. Org. Chem. 8 (1943) 417. [4] S.D. Faust, E.W. Mikulewicz, Water Res. 1 (1967) 509. [5] E. Emerson, I. Sagal, J. Org. Chem. 13 (1948) 535. [6] D. Svobodova, J. Gasparic, Coll. Czech. Chem. Commun. 35 (1970) 1567. [7] D. Svobodova, J. Gasparic, L. Novacova, Coll. Czech. Chem. Commun. 35 (1970) 31. [8] Y.C. Fiamegos, G.A. Pilidis, C.D. Stalikas, A.E. Dados, M.I. Karayannis, Fresenius Environ. Bull. 7 (1998) 558. [9] Y.C. Fiamegos, C.D. Stalikas, G.A. Pilidis, M.I. Karayannis, Anal. Chim. Acta 403 (2000) 315. [10] Y.C. Fiamegos, G.A. Pilidis, G. Varvounis, J. Heterocycl. Chem. 38 (2001) 1065. [11] P.F. Jones, K.E. Johnson, Can. J. Chem. 51 (1973) 3733. [12] V. Dave, J.B. Stothers, E.W. Warnholt, Can. J. Chem. 52 (1974) 2932. [13] R.J. Burton, K.E. Johnson, B.E. Robertson, Can. J. Chem. 65 (1987) 2082. [14] E. Emerson, J. Org. Chem. 3 (1938) 153.

4. Conclusions The six newly synthesized 4-aminopyrazolones when reacting with phenolics compound form colored quinonoid products. In all cases these products were analogous to the one formed by the similar reaction between 4-AAP and the phenolic compounds. The reaction with phenol itself is quantitative. Diaminopyrazolones (DAP, DADP) do not form diquinone-imide structures. The amine in the para