1962; “Trans. Sympoeium on Electrode Processes,” Chap. 5, p. 109, Wiley, Yew York, 1939. (9) Harris, IT. E., Kolthoff, I. )I., J . A m . Chem. SOC.68, llT5 (1‘146). (10) Kolthoff, I. h l . , Lingane, J. J., “PolarouaDhv.” Vol. I. 2nd ed.. Cham 13, Inteysrience, Sew York, 1952. (11) Kolthoff, I. >I., I’arrv, E. P., J . i l m . Chern. SOC. 73, .%15 (1951); ~
Sorhnik
Jfenzurad. Polarog.
Sjerdu
Pruzc., 1st Congr., 1951, Pt. 1, Proc.,
p. 145. (12) Lingane, J. J., J . A m . Chem. SOC. 61, 2099 (1939). (13) Lingane, J. J., Kerlinger, H., IND. E A G CIIEV., . k A L . ED. 13, 77 (1941). (14) Mark, H. B., Jr., Reilley, C. S . , J . Electroanul. Chem. 4, 189 (1962). (15) Xleites, L., “Polarographic Techniques,’’ pp. 75-82, Interscience, X e w York, 1955. (16) Ibad., p. 56.
(17) Tur’yan, Ya. I., Serova, G. F., Zhttr Fis. Khim 31, 1956, 2200 (1967). RECEIVED for review September 26, 1962. Accepted December 14, 1962. Research sponsored in part by the U. S.Air Force, Office of Scientific Research and Development Command, under Contract KO. 49(638)-333. Presented a t the Combined Meeting of the Southeast and Southwest Sections, .4CS, New Orleans, La., December 7-9,1961.
Comparison of Spectrophotometric and Spectrophotofluorometric Methods for the Determination of Malonaldehyde EUGENE SAWICKI, THOMAS W. STANLEY, and HENRY JOHNSON Roberf A. Taft Sanitary Engineering Center, Cincinnati 26, Ohio
b Ten new methods for the determination of malonaldehyde are compared with the thiobarbituric acid method. The reagents used in the new procedures are aniline, 4-hexylresorcinol, N-methylpyrrole, indole, 4’aminoacetophenone, ethyl p-aminobenzoate, 4,4’-sulfonyldianiline, pnitroaniline, and azulene. The most sensitive spectrophotometric method i s the thiobarbituric acid method; the most highly selective is the pnitroaniline method. The spectrophotofluorometric methods are b y f a r the most sensitive; they are highly selective also. Nanogram amounts of malonaldehyde can be determined with very little interference from other compounds. All methods measuring absorbance are in compliance with Beer’s law. The spectrophotofluorometric methods measuring emission show a similar linear relation between the emission reading and the concentration. Most of the methods have a reasonable preckion and color stability.
I
a continuation of prerious research on the origination of methods of analysis for ox) genated organic fragments that could contribute to air pollution, a group of diverse procedures for the detwmination of malonaldehyde are introduced. The availability of a group of methods giveq the researcher a distinct advantage in obtaining optimum results for a specific test mixture. Also, the results of analyses by several methods may be checked against each other. Nalonaldehyde exists in aqueous acidic solution mainly as the tautomer, 8-hydroxyacrolein (14). It can be determined in aqueous acidic (A,,, = N
245 nip, E = 13,000) or alkaline (A mar = 267 mp, E = 30,000) solutions. Obviously the presence of organic compounds that absorb in the ultraviolet region would interfere seriously in this determination. Phloroglucinol has been used to determine nialonaldehyde; a red dye is formed ( 6 ) . llalonaldehyde also can be determined with barbituric or thiobarbituric acid and its I,3-diphenyl and 1,3-diethyl derivatives; the chromogens obtained absorb at 486, 530, 537, and 540 mp, respectively (21). The red phloroglucinol chromogen is about one fifth af intensely colored as the red thiobarbituric acid dye; t h r yellow barbituric acid chromogen is almost as intense as the thiobarbituric acid dye, but requires over 100 minutes of heating for quantitative condensation ( 2 2 ) . Of the published methods for the determination of malonaldehyde, the most satisfactory appears to be the thiobarbituric acid method. 4 disadvantage, however, is that the thiobarbituric acid reaction as applied t o animal tissue gives color reactions M ith various compounds and therefore ir not specific ( I S ) . Some of the interfering colors obtained are due to the decomposition of the thiobarbituric acid nhen it is heated in the presence of acids or oxidizing agents (19). If the mixture being analyzed contains hydroperoxides, the decomposition is accelerated. Many types of mixtures submitted t o the thiobarbituric acid test have yielded a n appreciable amount of absorption in the 440- to 460-mfi region (3, 4,10, 25, 26). Compounds such as glycidaldehyde and glyceraldehyde form a yellow chromogen absorbing a t 466 nip (16). d large amount of this background absorption would interfere seriously with
the determination of malonaldehyde by thiobarbituric acid. Thiobarbituric acid has been used to determine malonaldehyde and its derivatives as derived from rancid foods (do), linolenic acid ( 7 ) , 2-deoxy sugars and galactal (22), 2-aminopyrimidine and sulfadiazine (17 ) , highly unsaturated fatty acids (9), sialic acids (259, p-formylpyruvic acid (24), 2keto-3-deoxyheptonic acid (8), and (2). 2-keto-3-deoxygluconic acid p-Nitroaniline has been used for the detection of malonaldehgde ( 5 ) . Following periodate oxidation, deoxy sugars and glycals n-ere distinguished from methylpentoses by the deep yellow zone which they gave when the chromatogram was sprayed m-ith a n acidic solution of p-nitroaniline. The yellow color is due t o the neutral dianil, I, X = NO2,or its salt. The procedurrs introduced for the determination of nialonaldehyde and malonaldehyde-yielding compounds are compared with the thiobarbituric acid method. EXPERIMENTAL
Reagents and Apparatus. dzulene, 10% aqueous tetra-n-propylammonium hydroxide, and 3-niethoxyacrolein dimethyl acetal were obtained from Distillation Products, Rochester, 1;. Y.; 4’-aminoacetophenone, p nitroaniline, indole, 4-hexylresorcinol, and malonaldehyde bis(dimethy1 acetal) from Laboratory Services, Cincinnati, Ohio; S-methylpyrrole, 4.4’-sulfonyIdianiline, and S-ethylcarbazole from Aldrich Chemical Co., ,Ililwaukee, \Vis. Most of the other compounds were obtained from these sources also. All chemicals were purified by crystallization to a constant melting point or by distillation. VOL. 35, NO. 2, FEBRUARY 1963
0
199
Malonaldehyde bis(dimethy1 acetal) was used as a standard in the determination of malonaldehyde. This compound has been recommended for this purpose, as i t is readily hydrolyzed t o malonaldehyde (18). The calculated results obtained in each procedure are based on the assumption t h a t a theoretical yield of malonaldehyde is obtained from the acetal. For all procedures the blank had t h e same composition as the analyzed solution, except for the absence of malonaldehyde bis(dimethyl acetal). A Cary Model 14 recording spectrophotometer (cells of 1-em. path length, 3-ml. volume) was used in the spectrophotometric work. The following settings were used with the AmincoBowman spectrophotofluorometer: sensitivity 50, meter multiplier 0.01, slit arrangement No. 2, and Dhototube R C A T y p e 1P21 (I).’ 4-Hexylresorcinol Procedure A. T o 2.5 ml. of aaueous test solution are added 0.1 nil. bf 25% 4-hexylresorcinol in glacial acetic acid a n d 2.5 ml. of
a mixture of trichloroacetic acid (100 grams of acid dissolved in 10 ml. of water) and concentrated hydrochloric acid (38%) (vol./vol. = 4/1). T h e entire mixture is heated on t h e boiling water bath for 10 minutes and allowed to stand a t room temperature for 15 minutes. A reading is then taken a t the wavelength maximum of 603 mp against the blank. The color intensity at 603 mp is stable for 10 minutes and then gradually increases. The various methods are compared in Table I. 4-Hexylresorcinol Procedure B. T o 2.5 ml. of 2-methoxyethanol test solution are added 0.1 ml. of 20% 4-hexylresorcinol in 2-methoxyethanol and 2.5 ml. of a mixture of trichloroacetic acid (100 grams of acid dissolved in 10 ml. of water) and concentrated hydrochloric acid (vol./vol. = 9/,l). The mixture is heated on the boiling water bath for 10 minutes and allon-ed to stand a t room temperature for 15 minutes. A reading is taken at the wavelength maximum of 603 mp against
Comparison of Methods for Determination of Malonaldehyde Color ProDet. sta- ceRel. Dil. Sen- libil- dure E x std. fac- sitiv- mit; i t j , time, Reagent ,A, 10-3 devSa tor ityb pg. min. min. 4-Hexylresorcinol, Proc. A 603 1 2 . 4 1.1 2 6 . 2 2.9 10 27 4-Hexylresorcinol, Proc. B 603 46.0 7.0 2 23. 0.78 30 2T Aniline 387 48.6 1 . 9 2 24.3 0.59 >480 6 Indole 550 60.0 2 . 8 10 6.0 1.2 10 9 4’-Aminoacetophenone, Proc. B 504 66.7 1 . 2 4 16.7 0.44 >30 6 580 74.6 2.0 3 24.9 0.29 >30 6 m-Nitroaniline. Proc. B .5.% 85.0 3 . 4 12 7 . 1 1.02 Od 2 h-Methylpyrrble 7oi 135.0 1 . 7 io 13 5 o 53 5 7 Azulene, Proc. A 15 7 4 35.5 0 20 702 142.0 7.7 rlzulene, Proc. B 30 24 3 52.7 0.14 Thiobarbituric acid8 530 158 Of 5 . 0 Based on 10 determinations. x 10-3 bSens. = ___ dil. factor’ c Total micrograms of malonaldehyde in test solution giving absorbance of 0.1 in 1-em. Table 1.
Q
ceii.
Fades gradually. Procedure of Waravdekar and Saslaw (29). f hmax532 mp, E = 153,000 has been reported (29). e
Table 11.
Comparison of Spectrophotofluorometric Methods for Determination of Malonaldehyde
Reagent Quininee 4,4’-Sulfonyldianiline Ethyl p-aminobenzoate
350 475 490 500 475 490 ~~
p-Aminobenzoic acid 4’-+4minoacetophenone
450 545
7.4
1 8.0
550
5.0
5.0
520
0.8
0.7
0.5
0.6
~
500 510 475
Instrument set a t emission wavelength maximum. b Instrument set at excitation wavelength maximum. 0 In 0.1N H&Oa. Used as standard of comparison. 0
200
ANALYTICAL CHEMISTRY
1
the blank. The color intensity a t 603 mp is stable for at least 30 minutes. Aromatic Amine Procedures. MONOCATIOKIC PROCEDURE A. Two milliliters of aqueous test solution and 2 ml. of 3% aniline in Z-methoxyethanol containing 5% b y volume of concentrated hydrochloric acid are heated in a boiling water b a t h for 3 minutes and then cooled under t h e tap. T h e absorbance is read a t 387 mp. The color intensity a t this wavelength is stable for a t least 8 hours. XONOANIOXIC PROCEDURE B. One milliliter of aqueous test solution and 1 ml. of 3% p-nitroaniline in Z-methoxyethanol containing 5% by volume of concentrated hydrochloric acid are heated in a boiling water bath for 3 minutes and then cooled under the tap. One milliliter of 40% methanolic benzyltrimethylammonium methoxide is added. The absorbance is read a t 580 mp against the blank. The color intensity at this wavelength is stable for a t least 30 minutes. 4’-Aminoacetophenone can be substituted for p-nitroaniline in the procedure. I n this case 2 ml. of alkali is used. The absorbance is read at 504 mp; the color intensity is stable for a t least 30 minutes. Spectrophotofluorometric Procedure C. T o 2 ml. of t h e dimethylformamide test solution was added 1 ml. of a dimethylformamide solution containing 1% of ethyl p-aminobenzoate and 1% (vol./vol.) of concentrated hydrochloric acid (38%). T h e mixture was heated 6 minutes on t h e boiling water bath and then cooled t o room temperature. One-half milliliter of 10% aqueous tetra-n-propylammonium hydroxide was added. Readings were taken at the emission wavelength maximum of 550 mp while exciting at the excitation wavelength maximum of 490 mp. The fluorescence intensity was stable for 30 minutes. Other aromatic amines can be substituted for ethyl p-aminobenzoate (Table 11). N-Methylpyrrole Procedure. T o 1 ml. of aqueous test solution is added 1 ml. of 70% aqueous perchloric acid followed b y 10 ml. of 0.5y0 N-methylpyrrole in methanol. T h e absorbance is read immediately at 558 mp. Indole Procedure. T o 1 ml. of aqueous test solution is added 1 ml. of a fresh solution of 2 7 , indole in sulfuric acid (sp. gr. 1.84). T h e mixture is allowed t o stand 7 minutes and then cooled under t h e tap. It is diluted t o 10 ml. with glacial acetic acid. -4s the color intensity is stable for only 10 minutes, the absorbance is read at 550 nip within 10 minutes. Azulene Procedures. PROCEDURE A. To 1 ml. of aqueous test solution is added 1 ml. of 0.5% azulene in glacial acetic acid, followed b y 1 ml. of 7001, aqueous perchloric acid. T h e mixture is heated on the water bath for 5 minutes, cooled t o room temperature, and then diluted to 10 ml. with glacial acetic acid. T h e absorbance is read at 700 to 702 mp within 5 minutes after dilution. PROCEDURE B. T o 1 ml. of aqueous test solution is added 2 ml. of 0.25%
azulene in glacial acetic acid, followed by 1 ml. of 70% aqueous Perchloric acid. The mixture is shaken, heated on the water bath for 5 minutes, and then cooled to room temperature. The absorbance is read a t 702 mpm The color intensity is stable for about 15 minutes. after which it very slowlv increases. Mechanisms. The following types of reactions are postulated as taking place in the different procedures.
I,
4'-aminoacetophenone, malonaldehyde could be determined as the neutral chromogen, I, = [A mBx = -3901, as the monocationic salt (A,,419), or as the monoanionic salt, 11, = (A maX504). The hydrochloride of I, X = Ac, was prepared by the Arylamine Procedure A. It decomposed a t a temperature greater than 300" C. It was insoluble in acetone and dimethylforma-
x
II: ,
X = Ac or NO,
X = A c or
NO,
J
\
OHC - CH2-CH0
Y m. The dianil derivative, I, X = Ac, mas prepared by Aromatic Amine Procedure A. The precipitated hydrochloride was suspended in acetone, treated with a slight excess of alkali followed by excess water. The precipitate was crystallized several times from boiling water. An approximately 807, yield of yellow cottony crystals (m.p. 216-18" cor.) was obtained. Calculated for ClsHI8N2O1: C, 74.5; H , 5.88; N, 9.15. Found: C, 74.4; H, 5.87; N, 9.05. This compound gave the following spectral bands in various solvents [listed in the order: solvent, wavelength maximum in mp (molar absorptivity)] : dimethylformamide, 308 (20,000), 390 dimethylformamide con(55,000) ; taining 1% (vol./vol.) of concentrated hydrochloric acid, 284 (13,000), 419 (77,000) ; dimethylformamide containing 2% of 10% aqueous tetraethylammonium hydroxide, 527 (89,000) ; the spectrophotometric analytical solvent mixture, 504 (82,000); and the spectrofluorometric analytical solvent mixture, 518 (100,000). The compound also has an intense orange fluorescence in alkaline dimethylformamide solution with excitation maxima a t 380 and 520 mp and an emission maximum a t 580 mp. From a comparison of the molar absorptivities obtained with the pure chromogen and in Spectrophotometric Procedure 13, it was evident that an approximately 807, jield of chromogen was obtained. From the absorption spectral data it can be seen that a-ith
500
mide and soluble in boiling water and boiling dimethylformamide. The hydrochloride crystals have a brilliant yellow fluorescence under ultraviolet light. The hydrochloride of the dianil derivative; I, X = KO*, was prepared by the Analytical Procedure. This yellow compound was insoluble in acetone and dimethylformamide. I t was readily decomposed by boiling solvents. Calculated for C16H13C1S404: C, 51.7; H, 3.73. Found: C, 52.2; H, 3.83. Solutions of this compound in 2-methoxyethanol and dimethylformamide, each containing 27, of a 10% aqueous tetraethylammonium hydroxide, gave bands at 578 and 627 mu, respectively. This compound had spectral characteristics identical t o those of the chromogen obtained in the analytical procedure. The solid has a brilliant orange fluorescence under ultraviolet light. From the analytical and spectral evidence it can be concluded that the chromogens obtained in Aromatic Amine Procedure A have a structure similar to I but with a proton on the aza nitrogen, while the chromogens obtained in Procedures B and C have structure 11,
600
700
Figure 1 . Spectrophotometric curves obtained in determination of malonaldehyde Final concentration of malonaldehyde 1.5
. _.-._.. .......
X 10 -6M
p-Nitroaniline Procedure B 4'-Aminoacetophenone Procedure B Azulene Procedure A
VOL. 35, NO. 2, FEBRUARY 1963
201
I*
0.8
1.c
I1
I il
I I
A 0.5
Figure 3. Spectrophotofluorometric curves obtained in determinaiion of malonaldehyde A.
-Excitation - - - spectra 4’-Aminoacetophenone a t emission wovelength 5 8 0 m p -.. . , . .
SO0
600
700
Figure 2. Spectrophotometric curves obtained in determination of malonaldehyde
--. . . . .. -.
----
.. .. ..
X,mjJ
Final concentration of malonaldehyde 1.5 Indole N-Ethylcarbazole N-Methylpyrrole .-. . 4-Hexylresorcinol Procedure A 4-Hexylresorcinol Procedure B
Ethyl p-aminobenzoate a t emission wavelength 5 5 0 m p 4,4‘-Sulfonyldianiline a t emission wavelength 5 4 5 m p B . Emission spectra 4’-Aminoacetophenone a t excitation wavelength 5 2 0 m p Ethyl p-aminobenzoate a t excitation wavelength 4 9 0 m p 4,4’-Sulfonyldianiline a t excitation wavelength 475 m p , Corrected values obtained by subtracting blank readings from all spectra Final concentrations of malonaldehyde, 5.7 X 1 Oe6Mwith 4’-aminoacetophenone, 5 . 7 X 1 O-’M with ethyl p-aminobenzoate, 2.86 X 1 O-’M with 4,4‘-sulfonyldianiline ,
X 10-‘M
-.-.-
The postulated structure of the chromogen obtained in the Azulene Procedure is based on the fact that the analogous monomethine (11) and pentamethine (12j dyes absorb at 618 and approximately 820 mn, respectively. Consequently the trimethine dye mould be expected to absorb near 700 mp. I n the Azulene Procedure the chromogen has been found t o absorb a t 702 mp, so that I11 has been assigned as the structure. Evidence has been given that the aliphatic aldehydes attack indole in the 3-position to give orange-red monomethine dyes (16) analogous to IV but with a shorter chain of conjugation. Because of the expected violet color obtained in the indole procedure and the above evidence, the structure of the chromogen is postulated as the trimethine dye (IVj. A trimethine dye is also poqtulated as the structure of the chromogen obtained in the -Y-Methylpyrrole Procedure. Wo worthm-hile clue to the structure of the blue chromogen from the reaction of 4-hexylresorcinol and malon202
ANALYTICAL CHEMISTRY
aldehyde or acrolein has been found. The spectral curves of the various chromogens obtained in the spectrophotometric procedures are sh0LT-n in Figures 1 and 2. DISCUSSION
4-Hexylresorcinol Procedures. The variables in Procedure A were investigated. Optimum intensities were obtained with 25% Chexylresorcinol, 10 to 25% concentrated hydrochloric acid in trichloroacetic acid, 10 to 30 minutes of heating a t looo, and a standing time of a t least 15 minutes. With increasing amounts of concentrated hydrochloric acid in trichloroacetic acid the shoulder a t 545 mp becomes a band of increasing intensity, while the main band a t 603 mu loses intensity. The absorbance obtained in the recommended procedure increased gradually with standing time-for example, in one typical analysis the absorbance after the recommended 1&minute standing period was 0.66. After additional standing
periods of 10, 30, and 60 minutes the readings had increased t o 0.67, 0.69, and 0.71, respectively. Oxidizing agents such as mercuric chloride and ferric chloride did not improve results. Beer’s law was obeyed from 2.9 (-4 = 0.1) to 58 pg. of nialonaldehvde. The main interferences in Procedure A were acrolein and 3-methoxyacrolein dimethyl acetal. The former gave a band a t 603 n ~ Fwith a molar absorptivity of 1800; the latter formed malonaldehyde and thus gave a band a t 603 mp with a molar absorptivity of 8000. 2-Aminopyrimidine and sulfadiazine gave negative results. Fiftymilligram quantities of the follol\-ing compounds gave colorless to yellow t o orange colors: formaldehyde, acetaldehyde, propionaldehyde, glyoxal, glyceraldehyde, crotonaldehyde, methacrolein, pyruvaldehgde, furfural, benzaldehyde, glycidaldehyde, glycidol, acetone, dihydroxyacetone, acetylacetone, and 3-buten-2-one. The variables in Procedure 13 were also investigated. Optimum results mere obtained with 12 to 267, 4hexyresorcinol, 10 to 14% concentrated hydrochloric acid in the trichloroacetic acid reagent, 10 to 15 minutes of heating a t loo”, and a standing time of a t
least 15 minutes. The absorbance obtained in the recommended procedure was stable for a t least 30 minutes. Beer’s law was obeyed from 0.78 t o 16 pg, of malonaldehyde. The main interferences in Procedure I3 were acrolein, 2-arninopyrimidine, and 3-methoxyacrolein dimethyl acetal, all of which gave a band a t about 603 nip with molar absorptivities of 490, 280, and 30,000, respectively. Glycidaldehyde gave a band a t 540 mu with a molar absorptivity of about 500. l’he remainder of the compounds that gave negative results in Procedure A also gave negative results in Procedure 1% * Aromatic Amine Procedures A and B. The variables in these procedures were investigated. Optimum intcnsities were obtained with 2 to 10% of aniline, p-nitroaniline, or 4’-aminoacetophenone. 1 to 10% of concentrated hydrochloric acid (vol./ vol.) in t h e reagent solution, a n d 3 minutes of hcating in a boiling water bath in Procedure A or B. Conditions n’ere adjusted so that the faintest colored blank and the best reproducibility were obtained mith optimum intensities. I n Procedure A various types of aromatic amines could be used as the reagent, For example, with aniline, S-phenyl-p-phenylenediamine, and 4’aminoacetol,henoiie the following wavelength niauima and molar absorptivities were obtained: 387 (48,600), 427 (27,200), and 413 (75,000), respectirely. K i t h aniline as the reagent the color intensity was stable for at least 2 hours; Beer’s law mas obeyed from 0.59 to 12 pg. The main interference in this procedure was 3-methoxyacrolein acetal and any yellon material present in the test solution or formed during the analysis. Other variables lvere investigated in Procedure B. Of the alkalies triedc’.g., benzyltrimethylammonium methoxide, tetraethylammonium hydroxide, and sodium hydroxide-the first gave somewhat higher intensities, especially when 2 nil. of the 40% methanolic solution \\as used. Beer’s law \vas obeyed from 0.29 t o 6 pg. of malonaltlehyde in p-Sitroaniliiie Procedure 13 and froin 0.44 to 9 fig. in 4’-Amino:icetophenone Procedure B. The main interference in p-Xitroaniline Procedure I3 n as 3-methoxy:wrol(in dimcthyl acrtal, which gave a band a t 580 m p nitli a molar absorptivity of 4600. Fifth milligrams of the following conipoundb gave yellow t o orange colors: formaldehyde, acetaldehyde, propionaldehyde, acrolein, glj-cidaldcliyde, glyoxal, succinaldehyde, benzaldehyde, furfural, crotonaldehyde, i~yruvaldchyde. acetone, dihydroxyacetone, 3-buten-2-one, 2,3-butanedione, rtcetylacetone, l-phenyl-l,3-butane-
dione, pyridine, 4-pyridylpyridinium chloride. 2-aminopyrimidine, and sulfadiazine. Some of the more reactive aldehydes gave the following results (compound, wavelength maximum, and molar absorptivity) : acrolein, 535, 478; glycidaldehyde 451, 982; and glyoxal, 457, 1000. h large contribution t o such weak intensities could be due to impurities present in the acrolein, etc. When reacted in the procedure, a n alcoholic solution of p-N,N-dimethylaminocinnamaldehyde ga! e a band at 560 mp with a molar absorptivity of 91,000. This aldehyde is not soluble in water, however, and therefore would not interfere in the recommended procedure. The main interferences in 4’-.hniliOacetophenone Procedure B were 3methoxyacrolein dimethyl acetal, acrolein, and 4-pyridylpyridinium chloride, which gave bands a t 504, 482, and 450 with molar absorptivities of 45,000, 7500, and 60,000, respectively. The remainder of the compounds that gave negative reactions in p-nitroaniline procedure gave negative results in this procedure. p-Phenylazoaniline (O.5y0 solution) mas tried as a reagent in pNitroaniline Procedure B and a wavelength maximum was obtained at 605 mp with a molar absorptivity of 46,000. Spectrophotofluorometric Procedure C. Investigation of the variables in the recommended procedure using ethyl p-aminobenzoate as reagent disclosed t h a t the presence of water or alcoholic solvents inhibited the formation of fluorescence. Optimum results were obtained with 1 to 3% reagent, lY0 concentrated hvdrochloric acid in the reagent solution, and 5 to 10 minutes of heating time. Twenty-nine per cent methanolic tetraethylammonium hydroxide or 10% aqueous tetraethylammonium hydroxide could be substituted for tetran-propylammoniuni hydroxide with only a slight decrease in sensitivity. Aqueous tetramethylammonium hydroxide could not be used because it gave a strong turbidity. The fluorescence intensity was stable for 30 minutes. A linear relationship was obtained between the concentration of aldehyde from 0.005 t o 0.17 pg. and the meter reading a t the emission wavelength maximum of 550 mp with the instrument set a t the excitation navelength maximum of 490 nip or betlyeen the same concentrations and the meter reading a t the excitation mvelength maximum of 490 nip xith the instrument set at the emission wavelength maximum of 550 mp. The relative standard deviation for 12 runs n as 8.07,. Other aromatic amines were tried in Spectrophotofluoronietric Procedure C (Table 11). The most sensitive
method for the determination of nialonaldehyde was the spectrophotofluorometric method using 4,4’-sulfonyldianiline as the reagent. I n this method a linear relationship between the concentration of malonaldehyde and the meter reading was obtained from 0.003 t o 0.15 pg, with the meter multiplier set a t 0.01. Honerer, n-hen the meter multiplier v a s set a t 0.001 the determination limit was decreased to 0.0003 pg, of malonaldehyde. This value compares favorably with the determination limit of 0.0125 pg. (absorbance = 0.060) reported for the thiobarbituric acid method ( 2 2 ) . I n the dianilinosulfone procedure the relative standard deviation for 11 runs was 3.247,. The fluorescence intensity was stable for 30 minutes. The following compounds gal e negative results in all the fluorescent methods: formaldehyde, acetaldehyde, glyoxal, propionaldehyde, acrolein, glycidaldehyde, glyceraldehyde, pyruvaldehyde, 1,3-dihydroxyacetone, glycidol, biacetyl, 2-aminopyrimidine, 4-pyridinecarboxaldehyde, benzaldehyde, and sulfadiazine. Positive results were obtained with 3-niethoxyacrolein dimethyl acetal, a malonaldehyde precursor. h’ - Methylpyrrole Procedure. Investigation of the variables in t h e recommended procedure disclosed t h a t optimum results were obtaincd with 0.5% reagent as compared to lower and higher concentrations, a 10-nil. volume of t h e reagent as compared t o lower or higher volumes, and 1 nil. of perchloric acid as compared t o lower or higher volumes of this acid and as cornpared to sulfuric or hydrochloric acids. The color intensity obtained fades fairly quickly. The absorbance was approximately halved in 15 minutes. The blank was colorless, giving an absorbance of about 0.01 a t 558 mp. Beer’s law was obeyed from 1 to 10 pg. of malonaldehyde. At higher concentrations there was a slight de!iation from the linear. Indole Procedure. Inveitigation of the variables in the reconiniended procedure disclosetl t h a t optimum results were obtained nit11 1 to 37, reagent, 1 nil. of reagent solution, and a reaction time of 7 t o 20 minutes. K a t e r , sulfuric acid (si). gr. 1.64), concentrated hydrochloric acid (38%), 2 - methoxyethanol, phosphoiic acid (857c), and glacial acetic acid were tried as diluting solvents; sulfuric and acetic acids g a ~ ethe highest intensities. Acetic acid was ])referred to sulfuric acid because of the much stronger corrosive effect of the latter on living tissue. ITithout the final dilution the blanks are much more colored and the molar absorptiT ities are decreased to some extent. H o ~ ~ e r e r , VOL. 35,
NO. 2, FEBRUARY 1963
203
by decreasing the concentration of reagent, the dilution factor could be improved, if necessary. When a 2% solution of the reagent in 2-methoxyethanol containing 3% of 70% perchloric acid was substituted for the sulfuric acid reagent, negative results were obtained \Tith an aqueous test solution containing malonaldehyde. When the procedure was further modified by using a 2-methoxyethanol test solution and heating it on the water bath for 10 minutes (instead of alloming it t o stand at room temperature for 7 minutes), poslitive results were obtained with malonaldehyde, as shoxn by two bands a t 488 and 538 mp with molar absorptivities of around 50,000. The color stability was investigated also. From an initial value of 0.93 the absorbance rose in 10, 15, 40, 60, and 120 minutes t o 0.94, 0.95, 0.96, 0.97, and 0.98, respectively. Beer’s law was obeyed from 1.2 to 25 pg. of malonaldehyde. The main interferences in the procedure were the malonaldehyde-yielding compounds, 3-methoxyacrolein dimethyl acetal and 2-aminopyrimidine, which gave bands a t 550 mp with molar absorptivities of 41,000 and 3300, respectively. Other compounds that gave weak bands a t shorter wavelengths were (compound, long wavelength maximum, and molar absorptivity) : formaldehyde, 450, 450; acrolein, 485, 1400; glyoxal, 485, 2200; and acetaldehyde, 485, 870. Kegative results were given by sulfadiazine, pyridine, benzaldehyde, and pyridylpyridinium chloride. Substitution of N-ethylcarbazole for indole in the procedure gaye a band at 590 n u with a molar absorptivity of about 34,000. This alternative procedure was not investigated further. Azulene Procedures. Variables in Procedure A were investigated. Optimum results were obtained with 0.5% reagent and 5 minutes of heating on the water bath. Instead of water, 2-mrthoxyethanol could be used as the test solvent. Water could not be substituted for acetic acid as t h e final diluent, for its use resulted in a cloudy solution, I n an investigation of the color stability the intensity increased from an initial absorbance of 1.84 to 1.87 in 30 minutes, Beer’s law was obeyed from 0.20 ( A = 0.1) t o 4 ug. of malonaldehyde. The main possible interferences in the determination of malonaldehyde are the malonaldehyde-releasing compounds, such as 3-methoxyacrolein dimethyl acetal and 2-aminopyrimidine, which react t o give long wavelength bands at about 702 mp with molar absorptivities of 93,000 and 2900, respectively. Another possible interference is 4-pyridinecarboxaldehyde, which gives bands at 716 and 637 mp with molar absorptivi-
204
ANALYTICAL CHEMISTRY
ties of 1400 and 2500, respectively. The aliphatic aldehydes reacted to some extent in the procedure to give bands absorbing a t a shorter wavelength. The following wavelength maxima and molar absorptivities were obtained for the aldehydes: formaldehyde, 623, 1400; acrolein, 617, 2300; glycidaldehyde, 589, 12,000; glyceraldehyde, 593, 9600; pyruvaldehyde, 614, 5500; glyoxal, 615, 18,000; acetaldehyde, 618, 1000; and propionaldehyde, 618, 800. Two ketones that react somewhat similarly are 2,3-butanedione and dihydroxyacetone, both giving a band a t 615 mp with molar absorptivities of 5500 and 9700, respectively. Very large amounts of these aldehydes or ketones would interfere in any determination of malonaldehyde. I n Procedure B optimum results are obtained with 0.25% reagent. I n all procedures the blank run against water has a sharp band at 615 mM with an absorbance of 0.4 to 0.7; a t 702 mfi the absorbance is approximately 0.2. The color intensity is stable for about 15 minutes. For all other variables optimum results are obtained as in Procedure A. Beer’s law is obeyed from 0.53 ( A = 0.1) t o 11 pg. of malonaldehyde. The interferences are about the same as in Procedure A. COMPARISON OF METHODS
I n all except the N-methylpyrrole and indole methods external heat was required. I n the indole method the heat of mixing was sufficient t o initiate the reaction. The N-methylpyrrole method involved the fewest steps and the shortest determination time. The 4-hexylresorcinol and thiobarbituric acid procedures required the longest times for completion of one run. Aromatic Amine Procedures B and C were the most highly selective methods investigated in this study. The only serious interference was 3-methoxyacrolein dimethyl acetal, which would not usually be found in mixtures for analysis. Aromatic Amine Procedures B and C and the N-met,hylpyrrole, indole, and azulene methods gave their characteristic spectra only with malonaldehyde and malonaldehyde-yielding compounds. &4croleinreacts t o a slight extent in the thiobarbituric acid procedure t o give spectral bands at 530 and 498 mp and a shoulder at 462 mM. These bands have a molar absorptivity of about 150 and could be derived from impurities in the freshly distilled acrolein. Acrolein is a weak definite interference in the 4-hexylresorcinol method, as it gives a spectrum identical t o t h a t obtained with malonaldehyde. Glyoxal reacts also with thiobarbituric acid to give very weak bands at 521 and 546 mp (E 280 and 350, respectively), while
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glycolaldehyde gives a very weak band at 512 mp. The malonaldehyde precursor, sulfadiazine, gives a positive reaction only in the thiobarbituric acid procedure; a molar absorptivity of 25,000 is obtained. The least selective reagent is azulene, for i t reacts readily with aliphatic alde100 hydes t o give bands (absorbing a t mp shorter wavelength than obtained with malonaldehyde) with molar absorptivities ranging from 140 t o 18,000. Indole reacts weakly with some of the aliphatic aldehydes and gives bands absorbing at a shorter wavelength than that obtained with malonaldehyde. I n the analysis of some particular mixtures where a yellow background color is found, absorption of the chromogen a t shorter wavelengths would mean more possibility of interference in the determination of malonaldehyde. This tvpe of interference has been reported for the thiobarbituric acid method (13) and could be expected for Aromatic Amine Procedure A, 4’-Aminoacetophenone Procedure €3, and the N-methylpyrrole and indole methods. The thiobarbituric acid method is the most sensitive spectrophotometric method for the determination of malonaldehyde. Azulene Procedure A is almost as sensitive. The spectrophotofluorometric methods are, by far, the most sensitive of all. Of these, the 4,4’-sulfonyldianiline procedure is approximately 40 times more sensitive than the highly sensitive thiobarbituric acid method. The least sensitive were 4-Hexylresorcinol Procedure A and the indole method. The sensitivity of the latter could be improved b y decreasing its fairly high dilution factor. Attempts t o decrease the determination limit by using proportionally smaller volumes and a smaller volume cell with an unchanged path length showed that in most cases the reproducibility suffered drastically. I n p-Kitroaniline and 4’-lminoacetophenone Procedures B a fivefold improvement in the determination limit can be obtained by using one-fifth volumes and a carefully positioned 0.5-ml., 1-em. rectangular cell (Pyrocell Manufacturing Co., New York), Under these conditions the per cent deviation rose to 7% (10 determinations) for both procedures, whereas the determination limits for the p-nitroaniline and 4’-aminoacetophenone procedures dropped to 0.06 and 0.09 pg., respectively. The most satisfactory reproducibilities were obtained with Aniline Procedure A, 4‘-Aminoacetophenone and pNitroaniline Procedures B, 4,4’-Sulfonyldianiline Procedure C, 4-Hexylresorcinol Procedure A, and Azulene Procedure B. Relatively speaking, the poorest reproducibilities were obtained with Azulene Procedure A, 4-Hexylresorcinol Procedure B, the P-thiobar-
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bituric acid method, and the ethyl paminobenzoate spectrophotofluorometric method. However, the reproducibility of all the methods is probably adequate for most purposes. All methods conformed to Beer’s law. The optimum color stability at the wavelength maximum was shown by the spectrophotofluorometric and thiobarbituric acid methods, Aniline Procedure A, and p-Sitroaniline and 4’-Aminoacetophenone Procedures B: the poorest color stability was shown by Azulene Procedure B. LITERATURE CITED
(1) Aiiierican Instrument Co., Instruc-
tions 3lanual 656. (2) Ashwell, G., Wahba, A. J., Hickman, J., Bzochem. et Biophys. Acta 30, 186
(1953). (3) Dox, -1.W.,Plaieance, G. P., J . Am. (‘hem. SOC.38, 2164 (1916).
(4) Dunkley, W. L., Jennings, W. G., J . Dairy Sci. 34, 1064 (1951). (5) Edward, J. T., Waldron, D. M., J. Chem. Soc., 1952,3631. (6) Fleury, P., Courtois, J., Hammam, W. C., LeDizet, L., Bull. Soc. Chim. France 1955. 1290. ( 7 ) Fujimaki, M., Odagiri, S., Nippon Nogeikagaku Kaishi 28,963 (1954). (8) Hurwitz, J., Weissbach, A., J . Biol. Chem. 234,710 (1959). (9) Jennings, W. G., Dunkley, K. L., Reiber, H. G., Food Res. 20, 1 (1955). ( I O ) Kenney, RI., Bassette, R., J . Dairy Sci. 42, 945 (1959). (11) Kirby, E. C., Reid, D. H., J . Chem. Soc., 1961, 1724. (12) Ibid., p. 3579. (13) Landucci, J. M., Pouradier, J., Durante. M.. Comnt. Rend. Soc. Biol. (14) Mashio, F., Kimura, Y., Nippon Kagaku Zasshi 81,434 (1961). (15) Patton, S . , Food Res. 25, 554 (1960). (16) Sawicki, E., “Microchemical Techniques,” Vol. If, N. Cheronis. ed.. Interscience, Kew York, 1962.
(17) Shepherd, R. G., ANALCHEM. 20, 1150 (1948). (18) Sinnhuber, R. O., Yu, T. C., Food Technol. IO, 9 (1958). (19) Tarladgis, B. G., Pearson, A. M., Dugan, L. Jr., J . Am. Oil Chemists’ Soc. 39, 34 (1962). (20) Tarladgis, B. G., K’atts, B. &I., Younathan, >I. T., Dugan, Jr., L., Ibzd., 37, 44 (1960). (21) Taufel, K., Zimmerman, R., Naturwissenschaften 6, 133 (1960). (22) Waravdekar, V. S., Saslaw, L. D., J . Biol.Chem. 234, 1945 (1959). (23) Warren, L., Ibid., 234, 1971 (1959). (24) Weissbach, A., Hurwitz, J., l b i d . , 234,705 (1959). (25) Wertheim, J. H., Procter, B. E., J . Dairy Sei. 39,391 (1956). 126) Wilbur. K. hI.. Bernheim. F.. ‘ Shapiro, 0.IT., Arch. Biochem. Riophys. 24, 305 (1949). I
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RECEIVEDfor review July 25, 1962. Accepted December 10, 1962. 144th Meeting, ACS, Los Angeles, Calif., March 1963.
Chronopotentiometry in Thin Layers of Solution CHARLES R. CHRISTENSEN and FRED C. ANSON California lnsfitufe o f Technology, Pasadena, Calif.
b The theory of chronopotentiometry of an electroactive species confined in a thin layer of solution next to the electrode is presented. The potentialtime and transition time relationships are verified for the reduction of iron(1ll) in 1F perchloric acid for solution layer thicknesses from 2 X 10-3 to 1 X 10-2 cm. The technique i s shown to be especially useful for studying irreversible systems that give poor diffusion chronopotentiograms and for determining the number of electrons transferred in electrochemical reactions. Application of the technique to the oxidation of N,N-dimethyl-pphenylenediamine in 1 F sulfuric acid i s demonstrated.
I
study ( I ) , electrodes inade by sealing platinum wire into the end of a length of soft glass tubing were occasionally defective in that the glass was not bonded to the wire over the entire area of the seal, and a layer of solution of an electroactive species could be trapped between the glass and metal. If the exposed surface of the electrode was washed free of reactant and the electrode was placed in a deaerated solution of supporting electrolyte containing no dissolved reactant, chronopotentiograms corresponding to the reactant trapped in a thin layer next to the electrode could be obtained (1-3). I n this investigation two types of thin layer chronopotentiometric electrodes have been devised for which the dimen-
sions of a layer of solution confined a t its surface are known. The equations have been derived for the transition time and for the electrode potential as a function of time for these electrodes. These relations were tested and verified for the reduction of iron(II1) in 1F perchloric acid. A comparison of the potential us. time curves for thin layer chronopotentiometry with those for diffusion chronopotentiometry of p-phenylenediamine and N,N-dimethyl-p-phenylenediamine has been made. The number of electrons involved in the oxidation of X,N-dimethyl-p-phenylenediamine in 1F sulfuric acid a t platinum electrodes mas determined to test the utility of the thin layer technique for the determination of n values.
K A PREVIOUS
EXPERIMENTAL
Apparatus. T w o different types of electrodes were designed for thin layer chronopotentiometry. Wire electrodes consisted of a length of 0.030-inch diameter platinum wire sealed in 5-mm. soft glass tubing b y heating in a flame (Figure 1 A ) . Before sealing, the wire was electroplated with copper t o a thickness of 1 to 1.5 X 10-3 em. for a distance of 1 em. from one end. The glass was fused around the wire t o cover 5 mm. of the copper-coated section and 5 mm. of the adjacent clean platinum. The copper was removed from the wire b y successive treatments with warm concentrated nitric and hydrochloric acids, and the wire protruding from the
glass \vas cut off as close as possible t o the end of the glass tube. A plate electrode was used in experime& where somewhat thicker layers of solution were desired. This electrode consisted of a 0.010-inch thick platinum disk 0.375 inch in diameter that was cemented Kith an epoxy resin (Resiweld Adhesive No. 4) into a hole in the face of a glass plate (Figure 1B). An insulated wire from the rear of the glass plate furnished the electrical connection. The electrode was held a uniform distance from a second glass plate by a 0.004-inch polyethylene gasket coated with silicone high vacuum grease. Two slits in the gasket, about 0.7 mm. wide, allorTed electrical contact from the working electrode to the auxiliary and reference electrodes in the external solution and permitted the cavity t o be filled with solution. Clamps held the two plates together. The working electrode used in diffusion chronopotentiometry &-asa piece of platinum foil, 0.4 sq. em. in area, welded to a platinum wire sealed in glass. A similar electrode with an area of 6.5 sq. cm. was used as the working electrode in the controlled potential electrolysis. The chronopotentiometric circuit and instrumentation have been described (1). A commercial potentiostat and current integrator from Analytical Instruments Inc., Bristol, Conn., was employed in the large scale controlled potential electrolysis. The temperature of all solutions was within 2’ of 24’ C. Potentials were measured with respect t o a calomel electrode saturated with sodium chloride but are reported vs. VOL. 35, NO. 2, FEBRUARY 1963
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