Mikrochimica Acta [Wien] 1977 I, 285--295

MIKROCHIMICA ACTA 9 by Springer-Verlag 1977

Faculty of Pharmacy, Charles University, 50027 Hradec Kr~ilo%, Czechoslovakia

On the Mechanism of the Colour Reaction of Phenols With 4-Dimethylaminoantipyrine

By D. Svobodovfi, M. Fraenkl, and J. Gasparie With 3 Figures

(Received

January 7, 1976)

In 1940 the highly sensitive colour reaction of phenols with 4-aminoantipyrine(I) was introduced into analytical practice by Emerson 1. It was stated by the same author that 4-dimethylaminoantipyrine(II) did not react 2 and that this reaction could give the possibility of the determination of I in the presence of II.
C6Hs

C6H5

/
CHa--N __

N CHa--N __

/
CH~

N /

CH3 ~ N H 2
I

\__/
II

N/'CH~ N, CH a

Later it was shown that this was not so: if the concentration of II was increased the same colour appeared under the same reaction conditions 3. T h e identity of the coloured products obtained by the reaction of phenols with I and II was proved 3-5. T h e same coloured product was also obtained when the reaction was carried out with 4-methylaminoantipyrine 4, 5 Several contributions have appeared describing the determination of phenols with II and an oxidizing agent. T h e reaction was carried out in ammoniacal buffer at p H 8--96-21. T h e reaction conditions were found empirically. T h e methods were evaluated as

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D. Svobodov{t et al.:

being equivalent 6-1~ to the original method according to Emerson, but in some cases their sensitivity was found to be higher ~1. In principle, two mechanisms for this reaction have been proposed. 1. Degradation of II to 4-aminoantipyrine by the action of the oxidizing agent, followed by reaction of the 4-aminoantipyrine with the phenol (this was not experimentally proved) 21. 2. T h e formation of a quinoneimine by the reaction of ammonia with the phenol and an oxidizing agent, and then reaction of the quinoneimine with II 5. In our laboratory we have investigated systematically the colour reactions of phenols in which they yield quinoneimine dyes. T h e reaction of phenols with both 4-aminoantipyrine 22 and I123 has been investigated in detail. In the present work we wish to contribute to the elucidation of the mechanism of the reaction of phenol with II.

Experimental
Reagents and Apparatus
4-Dimethylaminoantipyrine (Lachema), recrystallized from cyclohexane; 4-aminoantipyrine (Merck), recrystallized from benzene; phenol p. a. (Lachema); 4-acetylaminoantipyrine, prepared according to the literature24; 2,6-dichloroquinonechloroimine p. a. (Loba-Chemie); 2,6-dichlorophenolindophenol (Loba-Chemie); potassium ferricyanide p.a. (Reanal); BrittonRobinson-buffer was prepared according to the literature25; other chemicals used were of current analytical purity. Spectrophotometric measurements were made with a Spektromom 360 spectrophotometer (MOM, Budapest). For pH measurements a pH-meter OP-201/2 (Radelkis, Budapest) with a glass electrode and a saturated calomel electrode was used. Thinqayer chromatography was carried out on Silufol | precoated sheets (Kavalier, Votice, CSSR).

Methods Detection of indophenol in the reaction mixture phenol, quinonechloroimine and II. Britton-Robinson buffer (pH 8.0, 50 ml) was successively treated with 0.02M solutions of II, phenol and 2,6-dichloroquinonechloroimine in the amounts shown in Table I. Three minutes after mixing, the reaction mixtures were extracted with 5 ml of chloroform. T h e extracts were dried with anhydrous sodium sulphate and 2#1 applied onto the start of the thin-layer chromatogram. At the same time the corresponding standard compounds were applied and chromatographed. T h e mobile phase was

On the Mechanism of the Colour Reaction of Phenols

287

benzene-acetone (7:3). T h e spots of II and 2,6-dichloroquinonechloroimine were detected with UV light (254 nm) (Fig. 1).

Influence of the sequence of addition of reagents. To 30 ml of Britton-Robinson buffer (pH 7.9) 9 ml of 0.1M dimethylaminoantipyrine and 2 ml of 0.1 M potassium ferricyanide were added, followed by 5 ml of 2 x 10 4M phenol, and dilution to 50 ml with the buffer. T h e phenol was added at 0, 30, 60 or 120 min after preparation of the mixture, and the absorbance was measured 60 and 120 rain after the phenol addition, at 510 n m against the blank without phenol in 1-cm cuvettes (Fig. 2). Detection of 4-aminoantipyrine after oxidative degradation of II.
Sodium hydroxide solution (2M, 100 ml) was mixed with 5 ml of 1M a m m o n i u m chloride solution, 5 ml of 0.1M 4-dimethylaminoantipyrine, 5 ml of acetic anhydride and 10 ml of 0.5M potassium ferricyanide. T h e mixture was stirred for 30 min and extracted three times with 10-ml portions of chloroform. T h e combined extracts were dried with anhydrous sodium sulphate and evaporated under reduced pressure at room temperature to a volume of 2 ml; 2 #1 of this solution were applied onto the start of the thin-layer chromatogram and the development carried out with benzeneacetone (1 : 1) as mobile phase (Fig. 3).

Preparation of 4-acetylaminoantipyrine. To 30 ml of 2N sodium hydroxide 2 ml of 0.1M of aminoantipyrine and 3 ml of acetic anhydride were added. T h e mixture was stirred for 10 rain and then extracted with 5 ml of chloroform. T h e extract was dried with anhydrous sodium sulphate and 2 #1 of the solution were applied onto the start of the chromatogram. Acetylation of II was attempted in the same manner, but no reaction was observed. Preparation of the reaction product of 2,6-dichlorophenoI with 4-aminoantipyrine. In a 50-ml volumetric flask, 30 ml of BrittonRobinson buffer (pH 8.0--10.0) were treated with 5 ml of 2 x 10-4M 2,6-dichlorophenol, 2 ml of 0.02M 4-aminoantipyrine and 4 ml of 4 x 10-2M potassium ferricyanide. After mixing, the reaction solution was extracted with 5 ml of chloroform. T h e extract was dried with anhydrous sodium sulphate and applied onto the start of the thin-layer chromatogram.

Preparation of 2,6-dichlorophenolindophenol. To 300 ml of a saturated solution of sodium chloride, 0.01 mole of phenol and 0.01 mole of 2,6-dichloroquinonechloroimine in 30 ml of acetone were added. T h e sodium salt of 2,6-dichlorophenolindophenol thus

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D. Svobodovfi et al.:

formed was filtered off, washed with sodium chloride solution and dissolved in 500 ml of water. T h e solution was filtered and the free indophenol precipitated by passage of carbon dioxide. T h e indophenol was purified by repeated conversion into the sodium salt (calculated amount of sodium hydroxide) and back into free indophenol. T h e final product was a crystalline dark red powder. Results and Discussion T h e results of our investigation of the reaction of phenol with 4-dimethylaminoantipyrine and an oxidizing agent ~a have shown that the reaction studied takes place in practically quantitative yield in both the presence and absence of ammonia. In the absence of ammonia no quinoneimine can be formed and thus the reaction mechanism given in the literature 5 cannot be valide. Kawamura s
NHa n~KaFe(CN)6(or chloramine)+ /~--%-\__/ 0H CoH5
I

----~

[x

:o1

pH8.0 ------:~

/Nx~ CHa--N 0 / /~" CHa~ = / " N ~ = O

C6H6 N CHa N// N ~ 0

(1)

/CHa + X-- NXCH'~

CH~ ~

N/CH~

\CHa
did not detect quinoneimine as an intermediate product, but supposed it to be formed on the basis of the negative reaction of phenol, ammonia and an oxidizing agent (indophenol was not formed) and the positive reaction of II with 2,6-dibromoquinonechloroimine (a coloured product is formed). It can be deduced that if this supposition is correct then the quinoneimine formed by the reaction of phenol with the oxidizing agent in the presence of ammonia should react with II more easily than with phenol. We can disprove this supposition by an experiment arranged as shown in Table I. Thus, phenol, quinonechloroimine and II were simultaneously present in the reaction mixture in all experiments except experiment I. In all experiments except I there was no formation of a coloured product corresponding to the product of the reaction of II with quinonechloroimine, but indophenol was formed in con-

On the Mechanism of the Colour Reaction of Phenols

289

siderable yield. It is evident therefore that the reaction of quinonechloroimine with phenol to form indophenol is preferred.

O=O=N--CI q C>--OH OH~ O=O:N~-->--O- ( ) 2

In experiment I (Table I) when only quinonechloroimine and II were present in the reaction mixture, a slight amount of coloured product identical with the product of Emerson reaction was formed:

CIl e5 l O--~
N--C1 CHa--N CH3~ / N~ 0

(3)

CH3--N
~,

/N\9
/~/
\~./CH~

C6Hs I

,~na--

--*'\--CH3

In all cases a large amount of II remained unreacted while nearly all the quinonechloroimine was consumed by the reaction with
Table I Experiment Phenol" . . . . . . . . . . . . . . . . . . 2,6-Dichloroquinonechloroimine* . . . . . . . . . . . . 4- D i m e t h y l a m i n o a n t i p y r i n e * Q/P** . . . . . . . . . . . . . . . . . . . I -5.0 2.5 100/0 II 0.5 4.5 2.5 90/10 III 2.5 2.5 2.5
50/50

IV 4.5 0.5 2.5 10/90

* O.02M s o l u t i o n , ml "* Q/P = molar ratio of q u i n o n e c h l o r o i m i n eto p h e n o l

phenol (experiments II--IV, Table I, Fig. 1). In experiment I only a small amount of quinonechloroimine was consumed (Fig. 1). T h e possibility of the oxidative degradation of II to 4-aminoantipyrine has been denied in the literature 5, because 4-aminoantipyrine was not detected chromatographically in the reaction mixture after the action of the oxidizing agent on II. It is evident from our previous results 22 that 4-aminoantipyrine reacts with the oxidizing agent at a rate very close to that of the reaction with phenol, yielding a reaction product which does not react with phenol. This
Mikrochim.Acta 1977I13-4 19

290

D. Svobodovfiet al.:

is the reason why it is not possible to find it in the reaction products.

In fact, the 4-dimethylaminoantipyrine still reacting with phenol is

.................. "LO

o
t

0 0 o 0
~
0,5

0
'/ 2 3 4

0
5

0
6

Fig. 1. Proof of i n d o p h e n o lf o r m a ti o n in the reaction mixture phenol, quinonechloroimineand 4-dimethylaminoantipyrine 1, 4 - Di m et h y l am i n o a n t ip y r i n2,; coloured product prepared by the reaction of e 2,6-dichlorophenol with 4-aminoantipyrine and oxidizingagent; 3, 2,6-dichlorop h e n o l i n d o p h e n oprepared by the reaction of phenol with 2,6-dichloroquinonel c h l o r o i m i n e - identicalwith authenticsample;4, 2 , 6 - d i c h l o r o q u i n o n e c h l o r o i m i n e ; 5, chloroform extract of the reaction mixture of 4-dimethylaminoantipyrine and 2,6 -d i ch l o r o q u i n o n e c h lo r o i m in e (experimentI in Table I); 6, chloroform extracts of the reaction mixtures of 4-dimethylaminoantipyrine, 2,6-dichloroquinonechloroimineand phenol (experimentsII--IV in Table I, results identical) Colour of spots: 9 red, @ p i n k , 9 colourless. Detection: UV light (254 nm)

Q~
( A

--@ 2

-<3 t

'

6b

'

4o

Fig. 2. The influence of the sequence of reagents addition on the reaction of phenol with 4-dimethylaminoantipyrine Concentrationof phenol = 2 x 1 0 - a M ; molar ratio phenol : II : oxidizingagent = i : 900 : 200; buffer p H 7.9; t = time of the action of the oxidizingagent on II. Absorbance measured at 510 nm (1-cm cuvettes) against a blank (1) 60 m i n and (2) 120 rain after the addition of phenol

On the Mechanism of the Colour Reaction of Phenols

291

2 hr after addition of the oxidizing agent. This can be considered, as an indirect proof that 4-aminoantipyrine is the intermediate under the reaction conditions (Fig. 2). T h e reaction was carried out in the absence of a m m o n i u m ions and therefore the quinoneimine cannot be formed. T h e assumption of Kawamura that the reaction proceeds via the formation of a quinoneimine species and oxidative splitting of the dimethylamino group of II could not be confirmed, because unsubstituted antipyrine does not react with 2,6-dichloroquinoneimine. T h e formation of 4-aminoantipyrine by oxidative degradation of II has been proved by carrying out the oxidative reaction in the presence of acetic anhydride in the reaction mixture. Any 4-amino-

...................
@ @
0

1,0
RF
05

@ @ o
I 2 3 .4 0

Fig. 3. Proof of formation of 4-aminoantipyrine by oxidative degradation of 4-dimethylaminoantipyrine with potassium ferricyanide 1, 4-aminoantipyrine; 2, 4-dimethylaminoantipyrine; 3, 4-acetylaminoantipyrine; 4, chloroform extract of the reaction mixture of 4-dimethylaminoantipyrine with acetic anhydride and potassium ferricyanide. Detection: UV light (254 nm)

antipyrine formed was immediately converted into 4-acetylaminoantipyrine which is stable under these conditions and was identified chromatographically by comparison with an authentic sample (Fig. 3) :

C6H~
I

C6H~
I

N C H 3 -a ~ //N~NNN~OCHa CH -N /~ / CH3

K3Fe(CN)6 (CHaCO)20 -~

/ n ~ ~O CH3~ )/ CHa--N N / H' / NOC--CHa

(4)

4-Dimethylaminoantipyrine is a pharmaceutically important compound and therefore the mechanism of its oxidative degradation in the organism has been investigated 26-34. T h e results of our study

292

D. Svobodovfi et al.:

are in agreement with the conclusions of the investigation of the action of mild oxidizing agents on itas,a6; in nearly all cases 4aminoantipyrine has been found as an intermediate of the oxidative degradation. Oxidative degradation of 4-dimethylaminoantipyrine proceeds according to ref. 36 following scheme 5.
C6Hs C6Hs I --~ CHa-- N CHa~ )// N/H '~CHa C~H5 I N "~ / ~ CHa ~---~ C6Hs I CHa -- N CHa~NH~

/NI
CHa~ = _

1.

N( Gila CHa

2.
C6Hs I j N Cna__ F ~ CHa-

3.

02 (--NHa) light

O NH

(5)

4. 1, 4-dimethylaminoantipyrine, 2. 4-methylaminoantipyrine, 3. 4-aminoantipyrine, 4. imino-bis-phenazone From these results we propose the formation of 4-aminoantipyrine as the intermediate in the reaction of II with phenol both in the presence and absence of a m m o n i u m ions (their presence does not change the chromatographic and spectral results obtained) or affect the influence of factors such as pH, concentration of reagents etc., but does change the reaction rate). T h e presence of a m m o n i u m ions may inhibit a further oxidative degradation of II according to the following Scheme 6:
C~Hs I CHa-CH8 ~ 1. / NH2 <--~-~ C~H~ I CHa--N CHa-~ ~O NH 2. ON~ ~ C6H5 I -- CHa + NHa CHa

(6)

1. 4-aminoantipyrine, 2. imino-bis-phenazone This may be the reason why it is possible to obtain a quantitative yield of the coloured reaction product in the reaction with phenol at a lower concentration of II in the presence of a m m o n i u m ions

On the Mechanism of the Colour Reaction of Phenols

293

than in their absence 2a. T h e Emerson reaction 7 itself is not influenced by a m m o n i u m ions 22 (Scheme 7).
C~H5 I / NN CH3--N \/ / ~AO + ~ O H CHa~'~NH~ C~H5 I N +4OH- ~ CH~--N \ ~ O /
~ 4 e

(7)

1.

N= ~ - - O 2. 1. 4-aminoantipyrine, 2. antipyrylquinoneimine

CHa~ = ~

Summary

On the Mechanism

o[ the Colour Reaction 4- Dimethylaminoantipyrine

of Phenols With

In connection with a systematic investigation of the colour reaction of phenols to form quinoneimine dyes the reaction of phenol with 4-dimethylaminoantipyrine and an oxidizing agent has been studied. Though it has been found that the resulting coloured reaction product is identical with that of the reaction of phenol with 4-aminoantipyrine and an oxidizing agent (Emerson reaction), a different mechanism is operative. T h e reaction of phenol with 4-dimethylaminoantipyrine seems to proceed through oxidative degradation to 4-aminoantipyrine which reacts with phenol according to the Emerson reaction. In the literature this has been explained as occuring with intermediate formation of a quinoneimine, for which the presence of a m m o n i u m ions in the reaction mixture is necessary. In this communication the role of the a m m o n i u m ions is explained as inhibition of the decomposition of 4-dimethylaminoantipyrine to form imino-bis-phenazone. Phenol can also react with aminophenazone and an oxidizing agent in the absence of ammonium ions. Zusammenfassung Bei eingehender Untersuchung der Reaktion von Phenol mit 4-Dimethylaminoantipyrin ergab sich, daf~ das farbige Reaktionsprodukt mit dem der Reaktion yon Phenol mit 4-Aminoantipyrin und einem Oxydationsmittel (Emersonsche Reaktion) identisch ist, dag aber diese Umsetzung anderen Gesetzm~igigkeiten folgt. Die untersuchte Farbreaktion scheint so zu verlaufen, dag 4-Dimethylaminoantipyrin zun/ichst oxydativ zu 4-Aminoantipyrin abgebaut wird, das dann mit Phenol nach Emerson reagiert. In der Literatur wird der Reaktionsablauf durch die intermediiire Bildung von

294

D. Svobodovfi et al.:

Chinonimin erkl/irt, wozu aber die Anwesenheit von Ammoniumionen im Reaktionsgemisch erforderlich ist. Der positive Beitrag der Ammoniumionen wird jedoch damit erkl/irt, daf~ diese die Zersetzung yon 4-Dimethylaminoantipyrin verlangsamen. Schlief~lich verl/iuft die Umsetzung mit 4-Dimethylaminoantipyrin und Oxydationsmittel auch bei Abwesenheit von Ammoniumionen.

References
1 E. Emerson, U. S. Patent 2.194.201 (1940). 2 E. Emerson, J. Org. Chem. 8, 417 (1943). 3 j. Gaspari~, (;eskoslov. farm. 9, 514 (1960). 4 S. Ono, R. Onishi, M. Tange, K. Kawamura, and T. Imai, Yakugaku Zasshi 85, 245 (1965). K. Kawamura, Chem. Pharm. Bull. 16, 626 (1968). 6 V.T. Kaplin and N. G. Fesenko, Mater. Nau~n. Konferencii Sanit. Ochr. Vod. 1960, 118. 7 N. I. Amitina and A. M. Rosina, Koks i Chim. 1961 (4), 46. 8 V. T. Kaplin and N. G. Fesenko, Sovrem. Metody Analiza Prirodn. Vod., Akad. Nauk SSSR 1962, 136; Chem. Abstr. 58, 11961 f. (1963). 9 V. T. Kaplin and N. G. Fesenko, Zavodsk. Lab. 28, 187 (1962). l0 Z . M . Babe~kina, V.T. Kaplin and N. G. Fesenko, Gidrochim. Mater. 35, 207 (1963); Chem. Abstr. 59, 11100 f. (1963). 11 M.P. Babkin and A. L. Voloskovets, Ukr. Khim. Zhur. 30, 1347 (1964). le M.P. Babkin, Izv. Vyssch. Ucheb. Zaved., Khim. Tekhnol. 9} 270 (1966). ~a I. N. Oziganov and V. G. Martynenko, Koks i Chim. 1964, (2), 41. 14 V.T. Kaplin, S. E. Panchenko and N. G. Fesenko, Koks i Chim. 1964 (8), 41. 15 M. Csanady, Hidrol. Kozl. 44, 371 (1964); Chem. Abstr. 64, 4990b (1966). 16 V. D. Barskii and V. V. Noskov, Dokl. Vses. Soveshch. Plan. Eksp., 1st Moscow 1964, 270; Chem. Abstr. 68, 107912 r (1968). 17 V.V. Noskov and G.V. Semukhina, Khim. Produkty Koksovaniya Uglei Vostoka SSSR, Metody Analizov Vost. Nauchn.-Issled. Uglekhim. Inst., Sb. Statei No. 2, 64 (1964); Chem. Abstr. 62, 9799 d (1965). 18 V. V. Noskov and V. D. Barskii, Khim. Produkty Koksovaniya Uglei Vostoka SSSR, Metody Analizov Vost. Nauchn.-Issled. Uglekhim. Inst., Sb. Statei No. 3, 342 (1965). 19 V. D. Barskii and V. V. Noskov, Khim. Produkty Koksovaniya Uglei Vostoka SSSR, Metody Analizov Vost. Nauchn.-Issled. Uglekhim. Inst., Sb. Statei No. 3} 333 (1965); Chem. Abstr. 64, 11840 c (1966).

On the Mechanism of the Colour Reaction of Phenols

295

2o L. M. Alekseeva, Tr. Gos. Okeanogr. Inst. 113, 60 (1972); Chem. Abstr. 79, 9669 p (1973). 21 M. Pesez and L. Bartos, Ann. pharm. Fr. 25, 577 (1967). 22 D. Svobodov~i and J. Gaspari~, Coll. Czechoslov. Chemic. Commun. 33, 42 (1968). 23 D. Svobodovfi, J. Gaspari~, M. Fraenkl, and L. Novfikovfi, Coll. Czechoslov. Chemic. Commun. 41, 2176 (1976). 24 j. Ve~erkov~i, B. Kakfi4, B. Ve~erek, and K. K~icl, Pharmazie 21,

676 (1966). 25 V. S~kora and V. Zfitka, P~iru~nl tabulky pro chemiky, SNTL, Prague 1956. 26 F. Pechtold, Arzneimittel-Forsch. 14, 972 (1964). 2v M. Jaff4, Ber. dtsch, chem. Ges. 34, 2737 (1901). 28 M. Jaff6, Ber. dtsch, chem. Ges. 35, 2891 (1902). 29 j. Halberkann and F. Fretwurst, Z. physiol. Chem. 284, 97 (1950). 3o F. Pr6scher, Ber. dtsch, chem. Ges. 35, 1436 (1902). 31 j. Ve~erkov~i, B. Ve~erek, and E. Novotnfi, Ceskoslov. farm. 15, 491 (1966). 32 j. Ve~erkovfi, B. Ve~erek, and K. Kficl, Pharmazie 21, 682 (1966). 33 j. Ve~erkov~i, B. Kakfid, B. Ve~erek, and M. Ledvina, Pharmazie 22, 30 (1967). 34 j. Ve~erkov~i, J. Kohlidek, and K. Kficl, Pharmazie 17, 394 (1962). 35 H. J. Kienert, Diss. Braunschweig 1962; according to 36 86 F. Pechtold, Arzneimittel-Forsch. 14, 258 (1964). Correspondence and reprints: Dr. D. Svobodovfi, Faculty of Pharmacy, Charles University, 500 27 Hradec Krfilovd, CSSR.