I n Figurc 1, the distribution coefficients of several metal ions are plotted as a function of loading using 6JI nitric acid. The distribution coefficients show large variations with loading for thorium but only minor changes for yttrium and lead. I n column separations with 10-nil. samples containing 0.25 mmole of thorium plus 0.25 mmole of another metal ion, brmkthrough of the thorium was premature. By increasing the sample volume to 15 ml., this difficulty was avoided. If samples are used which cont.a.iii more than 0.25 mmole of thorium, the volume of loading solution should bc increased accordingly. However, excessive dilution of the sample solution ca,uses tailing of some elements separated from thorium and requires more fluent for their complete elution. The effect of common anions n-as in-
vestigated briefly. Addition of 2 ml. of concentrated sulfuric acid t o a sample containing 0.25 mmole each of thorium(1V) and uranium(V1) in 15 ml. of 6M nitric acid causes no interference in the quantitative separation and determination of thorium and uranium. Perchloric acid added to a thorium sample causes the thorium to break through too early unless most of the perchloric acid is removed by evaporating the sample almost to dryness. Chloride can be removed by evaporation; fluoride interference can be avoided by evaporation t o fumes of sulfuric or perchloric acid. Concentrated phosphoric acid added t o a thorium sample results in very incomplete removal of thorium from the anion exchange column by the usual 0.5M nitric acid stripping solution.
LITERATURE CITED
(1) Carswell, D. J., J . Inorg. AVucl,Chem. 3, 384 (1957). ( 2 ) Danon, J., J . Am. Chem. SOC.78, 5953 (1956). ( 3 ) Danon, J., J. Inorg. Nucl. Chem. 5, 237 (1958). ( 4 ) Fritz, J. S., Abbink, J. E., ANAL. CHEM.33, 1381 (1961). ( 5 ) Ichikawa. F.. Bull. Chem. SOC.JaDan ' 34,952 (196l).' ( 6 ) Korkisch, J., University of Vienna,
Austria, private communication, April
1962.
( 7 ) Korkisch, J., Tera, F., ANAL.CHEM. 33, 1264 (1961). ( 8 ) Nelson. F.. Kraus. K. A..J. Am. Chem. ' Boc. 76, $916 (i954j. ( 9 ) Tera, F., Korkisch, J., Hecht, F., J . Inorg. Nucl. Chem. 16, 345 (1961).
RECEIVEDfor review April 2, 1962. Accepted August 14, 1962. Contribution No. 1135 from the Ames Laboratory of the U. S. Atomic Energy Commission.
Spectrophotometric Determination of Trace Quantities of Alpha-Dicarbonyl- and Quinone-Type Conjugated Dicarbonyl Compounds D.
P. JOHNSON,
F. E. CRITCHFIELD, and J. E. RUCH
Research and Development Department, Union Carbide Chemicals Co., South Charleston,
b Microgram quantities of a- and conjugated dicarbonyl compounds are determined b y formation of the bis(2,4dinitrophenylhydrazones) and reaction of the latter with diethanolamine and pyridine to produce characteristic colors. The color due to excess 2,4dinitrophenylhydrazine reagent is eliminated b y treating the reaction mixture with 2,4-pentanedione which forms a colorless pyrozole. Monofunctional and nonconjugated difunctional carbonyl compounds do not produce colors under these conditions. Optimum reaction conditions and absorption maxima are presented for eight compounds.
I
(@, the authors described a method for determining low concentrations of lj4-benzoquinone by its reaction with 2,4-dinitrophenylhydrazine. The resulting bis-hydrazone was treated with a solution of diethanolamine in pyridine to produce a blue color which was measured spectrophotometrically. I n a continuation of that work, an investigation as made of the application of the same principle to t.he determination of other similar multifunctional carbonyl compounds. Of particular interest were 1,2naphthoquinone. lj4-naphthoquinone, anthraquinone. and other compounds N A PREVIOUS PUBLICATION
containing two or more carbonyl groups either adjacent or conjugated t o each other. The principle of bis(nitropheny1hydrazone) or osazone formation as a colorimetric means of analyzing a-dicarbonyl compounds has been covered rather extensively in the literature. Neuberg and Strauss (Sj reviewed the theoretical and practical aspects of the subject in 1945 and provided an excellent bibliography covering the period from 1874 to 1945. A year later the same authors (4) extended their discussion to quantitative formation of these products from various biological substances. Banks and associates (1) prepared the bis(2,4-dinitrophenylhydrazonejfor a colorimetric determination of glyoxal as an enzymatic oxidation product of carbohydrates. More recently, Sah-icki et al. reported the use of 4-nitrophenyll hydrazine and 2,4-dinitrophenylhydrazine for determining glyoxal by a similar principle (6). 2,4-Dinitrophenylhydrazine has been used by the present authors for some time for analyzing glyoxal ( 5 ) and other related dicarbonyl compounds ( 2 ) . I n their studies, Keuberg and Strauss used sodium ethylate as proton acceptor to produce the colored hydrazone species. Banks et al. employed alkaline acetone for the same purpose. I n both of these media, however, 2,4-dinitro-
W. Vu.
phenylhydrazones of simple monocarbonyl compounds such as acetone, and in general, all polyfunctional carbonyl compounds except 8-dicarbonyls likewise produce color. While the colors differ somewhat from those formed by the products of a-dicarbonyl compounds, they interfere in precise 'measurements of the latter substances. I n the present application, specificity is gained by using a mildly basic solution of diethanolamine in pyridine as the color developing medium. Under these conditions, only the 2,4-dinitrophenylhydrazones of vicinal and conjugated polyfunctional carbonyl compounds produce colors. EXPERIMENTAL
Reagents. Diethanolamine, 2% (v./v.) solution i n pyridine. 2,4-Dinitrophenylhydrazine. Dissolve 0.1 gram in 50 ml. of methanol a n d add 4 ml. of concentrated hydrochloric acid. Dilute t o 100 ml. with water and mix. 2,4-PentanedioneJ Union Carbide Chemicals Co. Procedure. Prepare a solution of t h e compound in t h e solvent specified in Table I. Pipet a n appropriate aliquot, not exceeding 3 ml., into a 25-ml. glass-stoppered graduated cylinder a n d add 1 ml. of 10% (v./v.) sulfuric acid. Add 1 ml. of 2,4dinitrophenylhydrazine reagent and VOL. 34, NO. 1 1 , OCTOBER 1962
1389
allow the mixture to react under the conditions specified in Table I. When the reaction is complete, add 2 drops of 2,4-pentanedioneJ mix well, and let stand 2 minutes. Cool to room temperature and dilute to 15 ml. with water. Add, with a pipet, 5 ml. of methylene chloride and shake well. Let the phases separate and pipet 3 ml. of the lower layer into a test tube. Evaporate the methylene chloride under vacuum a t room temperature and add. with a pipet, 10 ml. of the diethanolamine-pyridine reagent. Mix well and determine the absorbance of the solution with a spectrophotometer a t the appropriate wavelength, using 1-cm. cells. Use a reagent blank processed along with the sample to zero the instrument. Determine the concentration of the compound under test by applying the absorbance t o a previously prepared calibration curve. DISCUSSION
Compounds which were tested and found t o respond to this method are listed in Table I along with optimum reaction conditions and sensitivities of the method for the various compounds. Among the quinone type compounds listed are N,2,6-trichloro-p-quinoneimine and 2,6-dibromoquinone chlorimide, both of which hydrolyze in the reaction medium to form the corresponding halogenated benzoquinone which in turn reacts with 2,4-dinitrophenylhydrazine. Efforts to apply the method to anthraquinone and 2,3,5,6-tetrachlorop-benzoquinone were not successful, presumably because of lack of solubility of these compounds in the aqueous reaction medium. Among the vicinal dicarbonyl compounds listed in Table I are di-aldo, di-keto, and keto-aldehyde combinations. No special reaction or color characteristics were noted for any of these combinations. I t will be noted in Table I that different solvents are recommended for the various compounds. Wl--eas water is used for all vicinal dicarbonyl compounds and benzoquinone, methylene chloride is recommended for the naphthoquinones. W7ater solutions of the latter compounds are somewhat unstable, resulting in discoloration and decrease in concentration after short periods. Although aqueous solutions of benzoquinone likewise decompose slowly, water is nevertheless recommended as the solvent for this compound to avoid the two-phase reaction medium that would result with methylene chloride at the low reaction temperature. Absorption spectra of the bis(2,4dinitrophenylhydrasones) of glyoxal and 1,4-naphthoquinone, which represent the two classes of compounds studied, are shown in Figure 1. Glyoxal exhibits a n absorption maximum a t 580 mp while the 1,4-naphthoquinone de1390
0
ANALYTICAL CHEMISTRY
l. Compounds Analyzed by the Bis(2,4-dinitrophenyIhydrazone) Method Reaction conditions AbsorpTemp., Time, tion, Absorbance Compound Solvent "C. min. max., m r per pmole Water 25 30 615 3.24 1,4-Benzoquinone 640 2.23 Methylene 70 30 lJ2-Naphthoquinone chloride 645 2.94 70 30 Methylene 1,4Naphthoquinone chloride 580 1.70 Benzil Methylene 80 30 chloride 520 1.33 lJ2,3-Triketohydrindene Water 50 30 580 3.54 98 30 Glyoxal Water 575 2.52 Water 98 15 Pyruvic aldehyde 570 1.49 98 30 Diacetyl Water ... ... 615 ... NJ2,6-t~ichloro-p-quinoneimine ... ... ... 615 ... 2,6-Dibromoquinone chlorimide Table
.
rivative absorbed most strongly at 645 mp. Further inspection of Table I shows that in every case the a- or vicinal dicarbonyl products form winered colors which peak between 520 and 580 mp and that maximum absorption of all conjugated or quinone type dicarbonyl compounds occur in the blue region between 615 and 645 mp. 1,2,3-Triketohydrindene is unique in that it possesses structural characteristics of both classes of compounds. The carbonyl groups at the 1 and 3 positions are conjugated through the 8 and 9 carbons in a manner analogous to I, 4-naphthoquinone. The 1,2- and
I
.
2,3-dicarbonyl combinations, on the other hand, correspond to a-dicarbonyi compounds. Thus i t was expected that the tri~(2~4-dinitrophenylhydrazone) of lJ2,3-triketohydrindene would exhibit color characteristics of both structure types. Examination of Figure 2 shows that this is indeed true. Two distinct peaks are shown; one at 520 mp and another a t 620 mp. The peak at 520 mp possesses the greater extinction coefficient, probably because both the 1,2and 2,3-bis-hydrazone combinations contribute to its intensity. The color produced by treating a bis(2,4-dinitrophenylhydrazone) with a
0
1,4-?;?phth0quinone
1,2,3-Triketohydrindene
0
6
1
I
I 403
425
450
I
475 500 WAVELENGTH, m p
I 550
I
600
I 703
Figure 1. Absorption spectra of bis(2,4-dinitrophenyIhydrazones) of glyoxal and 1,4-naphthoquinone in diethanolamine-pyridine solution
Table II.
Effect of 2,4-Pentanedione Treatment on Recovery of Glyoxal and 1,4Benzoquinone
Recovered, fig. With Without pentanedione pentanedione 6.9 6.9 13.9 13.8 20.5 20.8 4.1 4.2 8.5 8.4 25.1 25.3
Added, pg. 6.9 13.8 20.7 4.2 8.4 25.2
Compound Glyoxal Glyoxal Glyoxal 1,4Benzoquinone 1,PBenzoquinone 1,PBenzoquinone
OIt
This reaction is characteristic of hydrazines and the enolic forms of p-dicarbony1 compounds. The decolorizing effect of 2,4-pentanedione on 2,4-dinitrophenylhydrazine was investigated as a means of eliminat-
base may be attributed to nitrogen deprotonation, causing electron shifts which result in a continuously conjugated ion. This principle is illustrated in Equation 2 using the 1,4-benzoquinone product as an example.
H
r\i 0
H
Table 111. Determination of Glyoxal in Monocarbonyl Compounds
NO2
2
”
I
I
NO2
NO2
0-
Colored Species ing excess reagent in the analysis of the a- and conjugated dicarbonyl compounds. Although the yellow color of the reagent does not interfere seriously in the analysis, it does obscure, to some degree, visual observations of low concentrations of the products under test, particularly those forming w-ine-red colors. Furthermore, the yellow background, in some cases, causes shifts in absorption maxima, resulting in calibration curves that deviate from Beer’s law. This effect was observed in the determination of 1,4-benzoquinone in acrylates ( 2 ) . To test the effectiveness of 2.4-pentanedione, two drops were added to the reaction mixture after completion of the hydrazone reaction. After standing for 2 minutes, the samples were processed for color. Data showing recoveries of glyoxal and 1,4-benzoquinone n.ith and without the pentanedione treatment are contained in Table 11. S o significant difference in the recoveries are shown; however, reagent blanks processed with pentanedione were Kater clear and remained so for approximately 30 minutes.
The colored product of 1, 4-benzoquinone thus is shown to contain three quinoidal moieties within the same conjugated system. a-Dicarbonyl products, on the other hand, contain only tvco quinoidal groups, but conjugation is sustained. The fact that the latter products possess one less double bond probably accounts for their absorption maxima occurring at lower wavelengths. The reactions of difunctional carbonyl compounds other than vicinal and conjugated structures were also investigated. Compounds tested included 2,4-pentanedione, 3-ketobutyraldehyde, adipaldehyde, and glutaraldehyde. Both adipaldehyde and glutaraldehyde formed bis (2,4-dinitrophenylhydrazones), but neither product produced color in the diethanolamine-pyridine solution. 2,4-Pentanedione and 3ketobutyraldehyde, lion ever, failed to produce hydrazones under the conditions of the method. Instead, both compounds reacted with 2,4-diriitrophenylhydrazine to produce colorless products The latter effect is due probably to pyrazole formation as illustrated in Equation 3 : H&
R I
c=o
I CH
C-OH ~
R
H-N
+
R
I
KO2
I
c=y
-
I
Figure 2. Absorption spectrum of tris(2,4-dinitrophenylhydrazone) of lf2,3-triketohydrindene in diethanolamine-pyridine solution
I
(3)
R I.
s
0 2
ReGlyoxal, % covery, y, Monocarbonyl Added Found 97.2 Acetaldehyde 0.36 0.35 Acetone 0.38 0.39 103.8 Acetone 0 76 0 75 98 7 2-Methylpentaldehyde 0 21 0 20 95 3 2-Methylpentaldehyde 0.42 0 41 97 7
With the exceptions of the chlorimides, all compounds in Table I were checked by this method for reproducibility and adherence to Beer’s law. All compounds tested responded satisfactorily. I n each case, the color remained stable for at least 30 minutes. Also, as shown by data in Table 111, the method was successfully applied to mixtures of glyoxal mith various monocarbonyl compounds. The lorrer limit of sensitivity for the latter application is governed by the ratio of 2,4-dinitrophenylhydrazine to the total reacting carbonyl groups in the sample. LITERATURE CITED
(1) Banks, T., Vaughn, C., Marshall,
L. M., ANAL.CHEM.27, 1348-9 (1955). (2) Johnson, D. P., Critchfield, F. E., Zbid., 33, 910-13 (1961). (3) Neuberg, C., Strauss, E., Arch. Biochem. 7, 211-30 (1945). (4)Zbid., 11, 457-65 (i946). (5) Ruch, J. E., Determinat,ion of
Glyoxal,” unpublished data, Union Carbide Chemicals Co. (6) Sawicki, E., Hauser, T. E., Wilson, R., ANAL.CHEX.34, 505-8 (1962).
RECEIVED for review June 4, 1962. Accepted July 26, 1962. Division of Analytical Chemistry, 142nd Meeting, ACS, Atlantic City, N. J., September 1962. VOL 34, NO. 1 1 , OCTOBER 1962
1391
