averaged 104%, W r was added to aliquots of serum (2 ml) and to liver homogenate aliquots (0.9 g), processed as described in the experimental section, and 51Cr recovery determined. These recoveries of 51Crfrom tissues averaged 95 %. Since the procedure of four washings with 1 N NaOH was rather rigorous, a standard solution of crystallized Cr(TFA)3 was carried through the clean-up procedure to determine any losses. No loss of Cr(TFA)3 was detected within the accuracy of the method. The original valence state of the Cr to be analyzed does not affect the analysis. Standards of either Cr(V1) or Cu(II1) were carried through the complete analysis and found to give identical results. To determine if species of chromium chelates other than Cr(TFA)3 were being formed in the analysis process, 51Cr which had been carried through the complete analysis was analyzed on a GLC. The effluent stream was split between a continuous flow radiomonitor and a flame ionization detector. Only one pair of radioactive peaks was seen and the retention times of these peaks corresponded to those of purified synthetic Cr(TFA)3. The combination of the four NaOH washes together with use of a 10-ft 3 OV-225 column produced a relatively clean chromatogram as seen in Figure 1. N o peaks overlapping the Cr(TFA)a trans isomer peak were seen. Other unidentified peaks did appear occasionally but these varied from sample to sample and never presented an analytical problem. DISCUSSION
The methods for Cr analysis in serum, blood, and urine recently published by J. Savory et al. (9, IO) and L. Hansen et al. (11) utilize the high resolution of gas-liquid chromatography to achieve specificity for Cr by separating its chelates from the other metal chelates present. They also both utilize the great sensitivity of the GLC to make possible detection of the extremely small quantities of chromium present in these tissues. The analysis of more complex biological tissues such as liver has proved a more difficult problem than blood, serum,
and urine. Liver contains more different metals and higher concentrations of metals than do the simpler tissues. In gas chromatographic analysis, many of these such as iron, copper, cobalt, and nickel, tend to emerge shortly after the solvent peak in large trailing peaks. These are probably thermal decomposition products and they obscure any Cr peaks present. Therefore, to analyze Cr in liver, it was necessary to improve resolution through more rigorous chemical purification of the chelation products and through GLC column materials with different retention properties. For the study of chromium tissue levels in physiological and deficiency states, it is necessary to measure Cr levels below 50 ppb (50 ng/g) This value had been the highest sensitivity achieved for tissue Cr using GLC methodology reported to date. The sensitivity for detection of pure Cr(TFA)3 standards is far in excess of this 50 ppb tissue Cr limit; however, the same sensitivity has not been possible for Cr contained in tissues. The method presented herein permits analysis of Cr concentrations in tissue of less than 20 ng Cr/g tissue. This permits the determination of levels of tissue Cr in physiological and deficiency states and, therefore, should provide a valuable tool for study of the metabolism of this element. ACKNOWLEDGMENT
C. Wetter and B. Fox were responsible for the GLC-MS determinations. J. T. Watson assisted with the mass spectra interpretations. J. Coniglio and A. Schulert provided fruitful discussion and suggestions. RECEIVED for review October 2, 1970. Accepted March 2, 1971. From a thesis to be submitted to the Graduate Faculty of Vanderbilt University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry. This work was supported in part by USPHS Grant No. AM 05441 and Vivian B. Allan M.D.-Ph.D. Fellowship. The LKB GLC-MS was purchased from funds supplied by NSF Grant No. GU-2057. The Varian Aerograph GLC was purchased in part from funds supplied by USPHS Grant No. ES00267.
Assay of Phenols and Arylamines via Oxidative Coupling David N. Kramer and Lucio U. Tolentino Physical Research Laboratory, Research Laboratories, Edgewood Arsenal, Md. 21010 Assay procedures are described for phenols and arylamines involving oxidative coupling with N,N-dimethylp-phenylenediamine to form the highly colored indaniline and indamine dyes, respectively. A potassium ferricyanide-sodium dichromate solution was found to be the best reagent for the oxidation of N,N-dimethyl-pphenylenediamine. The optimum conditions for maximum dye formation were at pH 9.5 for phenols and pH 6.1 for arylamines at 25 O C . The phenols and molar arylamines are assayable in the 10-6 to range with a relative standard deviation of 0.01 to 0.02.
AMONG THE MANY VARIED methods reported and in use for the assay of phenolic and arylamines compounds, the oxidative coupling procedure, employing N,N-dialkyl-o-phenylenediamine to form the highly colored indaniline and indamine dyes has been cursorily explored. Morita and Kogure ( I ) reported (1) Y.Morita and Y . Kogure, Nippon Kagaku Zasshi, 86 (1) 82 (1965). C . A,, 63 14064h. 834
ANALYTICAL CHEMISTRY, VOL. 43, NO. 7, JUNE 1971
a procedure for the determination of phenylenediamine isomers involving an oxidative coupling with phenol or a-naphtho1 using potassium ferricyanide to produce the colored indamine. The procedure required an extraction with chloroform, Camber (2) determined ketosteroid salicyloylhydrazones by oxidative coupling of N N-diethyl-p-phenylenediamine using a periodate oxidant. He utilized potassium ferricyanide oxidant in the estimation of phenols. He also investigated the use of silver nitrate and mercuric chloride as oxidants. The indaniline dyes, which have importance in the photographic industry, have been thoroughly investigated with respect to the mechanism of their formation (3),chromogeni(2) B. Camber, Nature, 175, 1085 (1955). (3) P. W. Vittum and G . H. Brown, J . Amer. Chem. SOC.,68, 2235 (1946).
city, and method of production (4). The indamines, lacking technical importance because of their instability, have not been explored. The reaction to form indanilines may be conveniently carried out by oxidizing an alkaline solution of N,N-dialkyl-pphenylenediamine and phenols, and may be effected by various oxidizing agents : potassium dichromate, potassium ferricyanide, potassium permanganate, sodium hypochlorite, manganese dioxide, silver chloride, and lead dioxide (3). Vittum and Brown (5) also showed that a number of p-substituted phenols, where the substituents withdraw electrons or facilitate the formation of quinoidal form of the phenol, undergo the indaniline coupling with displacement of the p substituent. We have employed an oxidative coupling system, potassium ferricyanide-potassium dichromate, to analyze, with a high degree of precision and sensitivity, solutions containing phenols and arylamines. Indeed, the reaction may be applicable with respect to any molecule which is capable of participating in the oxidative coupling reaction, such as barbituric acid, indole, and pyrazalone, although further studies are required. The reaction involved is as proposed by Vittum and Brown (3) (Scheme 1). A similar series of reactions may be written SCHEME
00
1
h
-
-
for the formation of indamines via arylamines. EXPERIMENTAL Apparatus. All recordings of spectra and absorbance were made using a Beckman Model DB spectrophotometer and recorder. A constant temperature bath was fitted to the spectrophotometer and all readings were obtained at 30" f 0.5 "C. Reagents. Dichromate-Ferricyanide Solutions (alkaline, for phenols): 4.47 g sodium dichromate dihydrate, 4.84 g potassium ferricyanide, and 4.0 g sodium hydroxide pellets were dissolved in 100 ml of distilled water to serve as a 0.15M stock solution of Na2Cr20i, and K3Fe(CN)6. The stock solution was diluted by adding 1 ml of stock to 49 ml of distilled water to make 0.003M solution. Dichromate-Ferricyanide Solution (neutral, for arylamines): 0.3348 g of potassium ferricyanide and 0.3030 g of sodium dichromate dihydrate in 200 ml of distilled water served as a stock solutidn 1.01 X 10-3M NazCrz07 and K?Fe(CNh. h,k-Dimethyl-p-phenylenediamine hydrochloride (Eastman Organic Chemical) 1.0 X 10-aM solution was prepared daily in glass distilled water and stored at 4 "C in a glass stoppered volumetric flask shielded from direct light. This was further diluted to 1.0 X 10-4Mfor the phenol assay. Buffers. Sodium carbonate buffer, O.lM, pH 9.5, was prepared in glass-distilled water. The procedure for the analysis of alkyl (or alkoxy-) substituted phenols: To a 10-ml volumetric flask there were added in the following order: 1 ml of 0.003M K3Fe(CN)6-K2Cr20i; 1 ml of 10- ' M solution of N,N-dimethyl-p-phenylenediamine hydrochloride; 1 ml of 0.1M sodium carbonate buffer pH (4) P. W. Vittum and C. H. Brown, J . Amer. Chem. SOC.,69, 152
(1947). (5) Zbid., 71, 2287 (1949).
1
io
IO
TIME
4b
3b
50
( Mln.)
Figure 1. Rate of dye formations Upper. 2 X 10-6Maniline Lower. 2 X 10-6Mphenol
Table I. Optimum Conditions Used in the Assay of Phenols and Arylamines ,,A Period of Compound (nm) reaction, pH of
to be analyzed Phenol 2,GDimethoxyphenol 2,6-Dimethylphenol Aniline N-Methylaniline N,N-Dimethylaniline
Dye
rnin
reaction
640
5 5
580
5
9.5 9.5 9.5
678 698 700
30 30 30
6.1 6.1 6.1
580
Buffer Carbonate Carbonate Carbonate Unbuffered Unbuffered Unbuffered
9.5; and 1.0 ml of phenol solution in the concentration range of 0 to 4 X 10e5M. The solution is then diluted to the mark and allowed to stand at room temperature for a period of 5 minutes (depending on the phenol). At the end of this period, the dye is read spectrophotometrically at the approximate wavelength against a blank. In the case of the phenol itself, the identical procedure was followed with the exception of the order of reagent addition which was as follows: 1. oxidizer; 2. phenol; 3. buffer; 4. diamine. The solution was allowed to stand 5 minutes. Procedure for the analysis of arylamines: To a 10-ml volumetric flask, there are added in the following order: solution (un1 ml of 1.01 X 10-3M K3Fe(CN)J)B-K2Cr207 buffered), 0-1.5 ml of the arylamines (depending on concentration), and 1 ml of 1.0 X 10-3M solution of N,N-dimethyl-p-phenylenediaminehydrochloride. The mixture is diluted to the mark and allowed to stand for 30 minutes. At the end of this period, the dye is read spectrophotometrically at the appropriate wavelength against a zero blank. RESULTS Determination of Phenols. Phenol determinations were performed according to the procedure described in the Experimental section. Calibration curves were obtained. Similarly, arylamine determinations were carried out as described and the calibration curves were obtained. The phenols and arylamines are readily assayable in the concentration range of 10-6 to molar, with a relative standard deviation of 0.01-0.02. Higher concentrations may be determined by suitable dilutions. Table I summarizes the optimum conditions found in assaying the phenols and arylamines. A determination was made of the rate of dye formation and its stability (Figure 1). In the case of phenol, the dye concentration reached a maximum in 5 minutes and was stable for 12 hours. On the other ANALYTICAL CHEMISTRY, VOL. 43, NO. 7, JUNE 1971
835
A
/
640,nm
0.4
0.3
02 0.I 0.0 -0.1
1
2 .o
1.0
x
3 .O
4.0
M Phenol
Figure 3. Comparison of oxidants in oxidative coupling of phenol with N,N-dimethyl-p-phenylenediamine 0.5
1.0
1.5
x
2.0
2.5
3,O
[Diamine] 0 [Dichromate] [Ferricyanide] A FerricyanideDichromate
M Aniline
Figure 2. Comparison of oxidants in oxidative coupling of aniline with N,N-dimethyl-p-phenylenediamine [Diamine] = 1.0 X lO-3M 0 [Ferricyanide] = 5.1 X lO-3M 0 [Dichromate] = 5.1 X lO-3M A FerricyanideDichromate = 2.5 X lO-3Meach
= = =
1.0 x i o - 4 ~ 4 5.0 X 10-3M 5.0 X 10-3M
=
2.5 X 10-3M
A 2.0
i-
Table 11. Effect of pH on Absorbance in Aniline and Phenol Assay
Final concentration aniline ( M ) 3 3 2 2
x
x x x
10-6 10-6
10-6 10-6 0 . 2 s x 10-6 0.25 x lo+
Absorbance
PH
0.69 0.83 0.49 0.59
7.2 6.1 7.2 6.1
0.045 0 . loo
7.2
0.140 0.150 0.153 0.155
7.83 9.5 9.85 9.95 9.75 9.99
Phenol ( M )
1x 1x 1x 1x 2 x 2
x
10-6 10-6 10-5
10-6 10-6 10-6
680
640
600
560
520
480
440
X nm
Figure 4. Spectra of indaniline dyes
0,305 0.340
hand, in the case of aniline, the maximum absorbance was reached at 30 minutes and the dye was also stable for 12 hours. Effect of pH. One ml of N,N-dimethyl-p-phenylenediamine, 10-3M, was added to a solution containing phenol or aniline, 1 ml of buffer, and 1 ml of oxidizing solution, then diluted to 10-mI volume. The solution was allowed to stand 0.5 hour and read spectrophotometrically. The absorbance readings are given in Table 11. Based on the results shown in Table 11, the arylamines were assayed at pH 6.1, and the phenols were determined at pH 9.5, with carbonate buffer. When the assay for phenols was determined at pH 6.1, the absorbance readings were identical with those obtained at pH 9.5; however, the rate of dye formation was slower by a factor of 8, requiring about 40 minutes to reach a maximum. Effect of Oxidizing Solutions. A study was made of the use of various oxidizing solutions to be employed in the oxidative coupling of the N,Ndimethyl-p-phenylenediamineand the arylamine. It was found that dichromate itself was almost as effective on an equivalent basis as dichromateferricyanide mixture. However, the use of ferricyanide alone was not satisfactory, Figure 2. 836
720
6.1
ANALYTICAL CHEMISTRY, VOL. 43, NO. 7, JUNE 1971
-
---
.. . ..
Phenol = 3.0 x 1 0 - 5 ~ 2,6-Dimethoxyphenol = 2.0 X 10-5M 2,6-Dimethylphenol = 2.0 X 10-5M
However, in the assay of phenol, it was found that the ferricyanide-dichromate combination was superior to either oxidant alone, as shown in Figure 3. Spectra of Indaniline Dyes. A study was made of the spectra of the indanilines formed upon oxidative coupling. Figure 4, presents the spectra obtained with phenol. The A,, of the indaniline curve was 640 nm at pH 9.5, carbonate buffer, after 30 minutes standing. The spectrum was identical with that of an authentic sample, whose reported A,, was 634 nm in 1 :1 acetone-water solvent (3). The indaniline sample obtained by oxidative coupling on standing 3 days revealed a spectrum whose A,, was shifted to 580 nm, indicative of a reaction occurring between the dye and reagent solution. A similar reaction occurred between the authentic indaniline and the buffer. The emaxof the authentic indaniline was 8.25 X lo3and that of the oxidatively coupled indaniline were identical. This is in variance with the reported emax = 2.8 X lo4 ( 3 ) . Similar results were obtained with the dye in aqueous medium, These results indicate that approximate quantitative conversion of the phenol to the indaniline was obtained. Spectra of Indamine Dyes. The spectra of the indamine dyes, obtained by the oxidative coupling process, are shown in Figure 5 (Scheme 2).
A
SCHEME 2
20
r
R
R
e
H N-'$=Nc*$,
om
H2N-zrN
o
N
R
2
H
DISCUSSION
The phenols and arylamines were determined in the lO-5M range. Higher concentrations may be assayed by appropriate dilution. Lower concentrations could be likewise measured by employing an extraction using chloroform or tert-butanol. The mechanism of the oxidative coupling of N,N-dialkyl-pphenylenediamine with phenols has been extensively studied by Tong and Glesmann (6). The rates were found to be proportional to the concentrations of quinonediamine and phenoxide ions, where the rate-determining step of the over-all reaction is the formation of the leuco dye by a bi-molecular reaction of the two ions, followed by a rapid oxidative conversion to the indaniline. The second order rate constant was pH-dependent, reaching a maximum in the pH region of 9-10. The predominating ionic species of the quinonediimine remains unchanged throughout this region, so that the rates are proportional to the phenol coupler ions (Scheme 3). In SCHEME d (dye) ~
~Kc i
d?
qulnonedllmide
IC1
LQDI':
3
, w h e r e LC-1 = p h e n o x i d e
and
CQDI'1
=
Ion.
the present study the pH for optimum color formation was 9.8, close to the pK, of the phenols. Also, similar to the observations of Tong and Glesmann ( 6 ) no induction periods were exhibited, which indicates no appreciable accumulation of leuco dyes in the course of the reaction. Vittum and Brown (3) have reviewed the synthetic and spectral aspects of the indaniline dyes, including an evaluation of the use of various oxidizing agents effective in the dye production. They elected to employ silver chloride as the oxidant and succeeded in preparing the dyes from N,N-dimethyl-p-phenylenediamine and variously substituted phenols. It was found in the present study that dichromate or a combination of dichromate-ferricyanide as oxidants was superior to ferricyanide alone in the assay of the arylamines, the latter effecting higher indamine dye formation within the period of assay. On the other hand, in the assay of phenols, the order of effective oxidative couplers was dichromate-ferricyanide > ferricyanide > dichromate. It has been reported that 2- or 3-substituted phenols readily underwent oxidative coupling whereas p-substituted phdnols such as p-cresol, p-hydroxyphenylacetic acid, p-hydroxybenzaldehyde, p-hydroxypropiophenone, ethyl-p-hydroxybenzoate, p-acetamino-phenol, hydroquinone, and p-hydroxyazobenzene showed little or no coupling (5). However, where the p-substituted was -C1, -Br, -C02H, -SO8H, -OR, -RCH-OH, R--0, dye formation was achieved ( 5 ) . It is reasonable to assume, that those phenols undergoing oxidative coupling are amenable to assay, but further analytical studies are required. In a limited study, arylamines have been detected qualitatively by their oxidative coupling with p-phenylenediamine to form indamine dyes (7), as shown in Scheme 4. The oxidant employed was persulfate. The spectra of the indamines are distinct from indanilines, ( 6 ) L. K. J. Tong and M. C. Glesmann, J . Amer. Chem. Soc., 79,
583 (1957). (7) 0. Heim, I d . Ejig. Chem., 7, 146 (1935).
I
760
720
680
560
600
640
520
A n rn
Figure 5. Spectra of indamine dyes __
--....
Aniline = 2.0 x 10-5714 N-Methylaniline = 2.0 X 10-6M N,N-Dimethylaniline = 1.5 X 10-5M SCHEME 4
+ 3H3 R
m
LBrownd,wski
H . Me
Bare)
Figure 4, and the arylamines may be distinguished from phenols by their formation of the indamine dyes. The instability of the indamine dyes is due in part to their susceptibility to hydrolysis. By performing the analysis at pH 6.1, the hydrolysis rate is appreciably reduced. The sequence summarizes the hydrolytic reactions (Scheme 5). SCHEME
5
01
220d
+
Re"
+
Ht
NH
Interferences. A determination was made of phenol and aniline in solutions containing albumin, 1 mg/ml. The phenol and aniline were assayed without interference by protein. The following ions were tested qualitatively at high concentrations for interference in the assay procedures and did not interfere : chloride, bromide, iodide, and cupric ions. Urea also did not affect the assay. Qualitative tests were positive for the following compounds using appropriate assay procedure : catechol, o-iodophenol, 3-cyanophenol, resorcinol, 2-aminopyridine, 3-hydroxypyridine, indole, and barbituric acid.
RECEIVED for review September 30,1970. Accepted February 15, 1971. ANALYTICAL CHEMISTRY, VOL. 43, NO. 7, JUNE 1971
837
