Rege neratio n of Ca rbo ny Is fro m hydrazones with Levulinic Acid
2,4- Dinitropheny I-
MARK KEENEY Dairy Departmenf, University of Maryland, College Park,
b Carbonyl compounds can b e regenerated from their 2,4-dinitrophenylhydrazones by heating with an excess of levulinic acid. This results in an interchange of levulinic acid for the carbonyl compound in the hydrazone. Dilute mineral acid is added to the levulinic acid to facilitate the regeneration of cr,$-unsaturated carbonyls. The procedure has proved useful for the regeneration of micro quantities of carbonyl compounds, making it particularly applicable to the study of flavor and odor compounds,
T
of 2,i-dinitrophenylhydrazine as a derivative reagent for carbonyl compounds has become very popular in recent years. The separation of unknown 2,i-dinitrophenylhydrazones by column (2, 13, 15, 16) or paper (6, 7 , 11) chromatography, followed by spectrophotometric observations ( 3 , 6 ) and possibly melting point determinations and elemental microanalyses, makes feasible the accurate identification of micro quantities (0 1 to 10 mg.) of hydrazones. It is often desirable. when working with hydrazones from natural systems, to regenerate free carbonyl compounds in order to study their chemical, physiological, or organoleptic properties. In this laboratory there was interest in regenerating niicro quantities of some carbonyls isolated from oxidized milk fat in order to study their organoleptic properties. There wenis to have been a general impression among users of 2,Pdinitrophenylhydrazine that the hydrazones prepared from this reagent are difficult to hydrolyze and that attempts to regenerate micro quantities of carbonyls would be impractical. However, a few workers have presented some specialized methods for regenerating the carbonyl compounds. Strain (16) suggested that treating the hydrazones with an escess of dicarbonyl compound, such as diacetyl (2,3-butanedione), in water, aqueous acids, or glacial acetic acid, would regenerate the parent carbonyl compounds. Anchel and Schoenheimer ( I ) reported that an excess of HE USE
Md.
pyruvic acid would split p-carboxyphenylhydrazones of saturated and a,@-unsaturated ketones. In later years, there was interest among steroid chemists in the regeneration problem because of the observations of Mattox and Kendall (9) that 2,4-dinitrophenylhydrazinewas an effective dehydrobromination reagent. Use of the hydrazine for this purpose created the problem of finding a method for regenerating the ketone-hydrazones formed as intermediates in the synthesis of steroids. hIattox and Kendall(9,lO) regenerated some steroids with pyruvic acid containing hydrobromic and glacial acetic acids. Djerassi (5) studied the hlattox-Kendall regeneration method and implied that water was necessary in the system in order to obtain effective regeneration. Demaecker and hlartin (4) regenerated steroid ketones by refluxing the hydrazones with acetone containing hydrochloric acid. Robinson (14) produced ketones in high yield by warming 2,4-dinitrophenylhydrazones with formic acid and copper carbonate. Consideration of the above reports suggested the possibility of developing a generalized reagent for regeneration of carbonyls from 2,4-dinitrophenylhydrazones. Factors necessary in a successful reagent would be carbonyl groups, acidity, and water. There was reluctance to use pyruvic acid as a source of carbonyl and acid groups because of the unstable ill-defined character of this compound. It was felt that levulinic acid would be a much more suitable compound. Study of the possibility yielded the following general observations: Saturated carbonyls were easily regenerated by heating levulinic acid solutions of 2,4-dinitrophenylhydrazones; conjugated unsaturated carbonyls (%enah, a,@-unsaturated ketones) were difficult to regenerate with levulinic acid. The addition of 10% water to the levulinic acid increased the regeneration rate of saturated carbonyls, but had little effect upon the unsaturated carbonyls; however, the addition of 10% 1.ON hydrochloric acid to the levulinic acid permitted the easy regeneration of both saturated and unsaturated carbonyls.
As a general practice, the use of about 50 weights of levulinic acid solution to 1 weight of hydrazone proved satisfactory. More levulinic acid solution may be used for compounds which are difficultly soluble in levulinic acid. Excellent regeneration of benzaldehytlc and cinnamaldehyde was obtained by using 200 weights of levulinic acid solution. REAGENTS
Levulinic Acid. SOLUTION A. Add 1 volume of water to 9 volumes of
melted levulinic acid. SOLUTIOX B. Add 1 volume of 1 .ON mineral acid (either hydrochloric or sulfuric) t o 9 volumes of levulinic acid. REGENERATION PROCEDURE
Place the 2,4-dinitrophenylhydraz1~iic in a test tube or distilling flask. Add 50 to 200 weights of levulinic acid solution. Use either Solution il or B, depending upon conditions. If the parent compound of the hydrazone is known to have no unsaturation conjugated with the carbonyl group, use Solution A. Use Solution B for hydrazones of conjugated unsaturated carbonyls. The sulfuric acid form of Solution B is recommended in cases where hydrochloric acid fumes may be objectionable, such as odor evaluation. Heat the mixture in a steam bath for 5 minutes. During the heating period, there will be a practically quantitative regeneration of a wide variety of carbonyl compounds (Figure 1). Remove from the bath after the S-minute heating period. At this point various procedures may be used t o study or isolate the regenerated carbonyl compound. If the regenerated carbonyl is a potent odor compound, the regenerated mixturr may be smelled directly or after dilution with water. I n some cases it may be desirable to steam distill the mixture to recover volatile carbonyls for further purification and study. If steam distillation is not desirable or applicable, the regenerated mixture may be extracted with petroleum ether, the extract washed with dilute sodium VOL. 29, NO. 10, OCTOBER 1957
1489
carbonate, and the ether evaporated to yield the carbonyl compound. DETERMINATION
OF REGENERATION RATES
General observations on the use of the levulinic acid procedure indicated that the regeneration gave a good yield of carbonyl compounds. For example, 200 mg. of heptanal 2,4-dinitrophenylhydrazone was placed in a 50-ml. distilling flask, together with 10 ml. of levulinic acid, Solution A. The flask was fitted with a delivery funnel and the side arm of the flask delivered into a Babcock skim milk test bottle. The flask was heated in a 100' C. oil bath for 5 minutes. Then 10 ml. of water was added to the flask and the temperature of the bath was rapidly raised to 150" C. About 2 ml. of aqueous distillate was collected in the cooled skim bottle. The bottle was removed from the apparatus and cold water was added to float the heptanal into the calibrated neck of the bottle. There was 0.068 ml. of heptanal in the neck, which represented a 70% recovery. The actual regeneration was greater than 70y0 because the steam distillation was not complete and the 50 ml. of water added to the Babcock bottle dissolved some of the aldehyde. In order to obtain a more accurate estimate of the rate and amount of regeneration of carbonyls from various classes of hydrazones, the data illustrated in Figure 1 were obtained. These data were calculated from spectrophotometric observations. The facts considered in this determination were: 2,4Dinitrophenylhydrazones have characteristic light absorption maxima and the molar absorptivity of any one class of hydrazones is practically a constant (3, 8) ; levulinic acid 2,4dinitrophenylhydrazone is slightly soluble in water, whereas 2,4dinitrophenylhydrazones of the carbonyls shown in Figure 1 are insoluble in water. It appears that the only 2,4-dinitrophenylhydrazones which have any appreciable solubility in water are polar-substituted compounds, such as keto-acids, or short chain aliphatics, such as acetone, acetaldehyde, or propionaldehyde The absorption maxima and molar absorptivity of the hydrazones used in this study were determined with a Beckman Model DU spectrophotometer. The values for chloroform solutions agreed closely with those in the literature (3, 8). There was a bathochromic shift of about 10 mb in the maxima of water solutions of hydrazones compared to chloroform solutions.
in the bottom of the tubes. Then 25 ml. of chloroform was added to one of the tubes in a series, and the absorbance of the solution a t the wave length of maximum light absorption for the particular hydrazone was determined. This gave an indication of the amount of hydrazone in each tube in a series. Two drops (about 50 mg.) of levulinic acid solution were added t o a tube containing the dry hydrazone deposit, and the tube rras immediately placed in a steam bath. After a specified period of time, the tube was removed from the bath and 25 ml. of water was poured into the tube. This added water stopped the regeneration reaction and dissolved the levulinic acid 2,4dinitrophenylhydrazone which had formed. The water solution was filtered if it was turbid. The absorbance of the water solution was determined a t 372 mp. By calculation from the absorbances of the chloroform and water solutions, the amount of levulinic acid 2,4-dinitrophenylhydrazone formed was determined and thus the per cent regeneration of carbonyl compounds deduced. Individual tubes from a particular series were heated for different periods of time to obtain the regeneration rate for each hydrazone illustrated in Figure 1.
Bishydrazones. a-Dicarbonyl compounds appear to be regenerated from bis(2,4-dinitrophenylhydrazones) a t about the same rate as a,&unsaturated carbonyls from monohydrazones. Exceptions are those bishydrazones which have a very low solubility in levulinic acid, such as 2,3-butanedione bis(2,4-dinitrophenylhydrazone). This compound proved difficult to regenerate because it required about 10 ml. of levulinic acid solution to dissolve 1.0 mg. of the bishydrazone When dealing with these
I
insoluble hydrazones, the regeneration efficiency can be improved by adding a solvent like nitrobenzene and a stronger mineral acid to the levulinic acid reagent. Quantitative regeneration of 2,3-butanedione was apparently obtained in 5 minutes a t 100" C. by the following procedure. One milligram of 2,3-butanedione bis(2,4-dinitrophenylhydrazone) was dissolved in 0.2 ml. of nitrobenzene a t 100' C., and 0.2 ml. of a solution composed of 9 volumes of levulinic acid and 1 volume of 5.ON sulfuric acid was added. The mixture was warmed in a steam bath and aliquots were removed each minute and tested for bishydrazones by the alcoholic sodium hydroxide test (12). During the heating period there was a gradual shift in the color produced in the alcoholic sodium hydroxide test from a distinct blue to a distinct red. This indicated a conversion of bishydrazones to monohydrazones (12). The actual formation of free 2,3-butanedione was confirmed by steam distilling a portion of the regenerated mixture and testing for 2 , s butanedione in the distillate. Reaction of the distillate with 2,4-dinitrophenylhydrazine yielded a hydrazone which gave a blue color in the alcoholic sodium hydroxide test. Substitution of 1.ON sulfuric acid for the 5.ON acid in the above procedure resulted in quantitative regeneration of 2,3-butanedione in 15 minut e s. Some unknown bi~(2~4-dinitrophenylhydrazones) of a-dicarbonyl compounds, which have been isolated in this laboratory from heated milk, were regenerated in less than 5 minutes with Solution B by the procedure used on monohydrazones. It appears that many bishydrazones can be regenerated by the procedure used on monohydra-
LEVULlNlC ACID
LEVULlNlC ACID-HCI
HEPTANAL- 0 2-HEPTANONE
CROTONALDEHYDE - 0 CINNAMALDEHYDE e
-
I
Stock solutions of the 2,Cdinitrophenylhydrazones in ethyl ether containing from 0.5 to 1 pmole of hydrazone per 10 ml. were prepared. Aliquots of 10 ml. of the ether solutions were transferred to a series of test tubes. The ether was carefully evaporated, leaving the dry deposits of hydrazones 1490
ANALYTICAL CHEMISTRY
-
5
IO
Left. Solution A
15 TIME
-
5 MINUTES
IO
I 15
Right. Solution B
Figure 1. Regeneration rates of carbonyl compounds from 2,4-dinitrophenylhydrazones at 100" C. Mole ratio of levulinic acid IO hydrazone, about 500 to 1
zones, and compounds which do not respond to the treatment can be regenerated by adding stronger mineral acid and a solubilizing agent such as nitrobenzene. APPLICATIONS
The fact that carbonyl compounds ranging in properties from those of simple aliphatic to highly conjugated aromatic (cinnamaldehyde) carbonyls were easily regenerated with levulinic acid-hydrochloric acid suggests that the method should have wide general application. The regeneration of 2,3butanedione from its bishydraxone suggests that the method may be extended t o conjugated dicarbonyl derivatives by tlie addition of various solvents. The ZJ4-dinitrophenylhydrazones of the carbonyl compounds formed in oxidized fat, such as saturated aldehydes, 2enals, and 2,4-dienals have been successfully regenerated. Spots of these fat derivatives, cut from paper chromatograms, yielded detectable odors when regenerated. The method has been useful in study of unknown carbonyl flavor compounds in dairy products. I n a study of the off-flavor compounds formed during the storage of dry milk, 2,4-dinitrophenylhydrazones were prepared from vacuum
steam distillates of the reconstituted milk. The hydrazones were separated by chromatographic procedures and then regenerated for organoleptic evaluation. The observations demonstrated the importance of carbonyls as contributors t o the characteristic flavor of dry milk and indicated which of the chromatographic fractions contained the most potent flavor compounds. Regeneration of the major flavor fraction and addition of the steam distilled carbonyl to fresh milk permitted a rough estimation of the flavor threshold of the fraction in milk. The estimate indicated that the reconstituted dry milk contained between 20 and 50 p.p.b. of the potent flavor carbonyl. Information on the flsvor potency is a valuable aid for identification purposes and of great value in indicating how much product must be worked up in order to obtain a desired amount of derivative. I n the case of the dry milk, it was estimated that approximately 300 pounds of powder mould have to be processed in order t o isolate l b t o 15 mg. of 2,4-dinitrophenylhydrazone of the major flavor fraction. LITERATURE CITED
(1) A4nchel, M., Schoenheimer, R., J . Biol. Chem. 114, 539 (1936).
Braddock, L. I., Garlow, K. Y . ,Grim, L. I., Kirkpatrick, A. F., Pease, S. )V., Pollard, A. J., Price, E. F., Reissmann, T. L., Rose, H. .2., Willard, M. L., ANAL. CHEY.25, 301 (1953).
Braude, F. .4., Jones, E. R. H , J . Chem. SOC.1945, 498.
Demaecker. J., Martin, R. H , Nature 173, 266 (1954).
Djerassi, C., J . A m . Chem. SOC.71, 1003 (1949).
Forss, D. -1.,Dunstone, E. .4 , Stark, IT., Chern. & Ind. 1954, 1292.
Heulin, F. E., Australzan J . Sci Research 5B, 328 (1052).
Jones, L. A , , Holmes, J. C., Seliyman. R. B., ASAL. CHERZ.28, 191 (1956).
Mattox, V. R., Kendall, E. C.,
.I. Am. Chern. SOC.70. 8S2 (1948). Ibid., 72, 2290 (1950). ' Meister, A , , Abendschein, P. .I., h A L . CHERf.
28, 171 (1956).
Neuberg, C., Strauss, E., Arch. Bzochem. 7, 211 (1945).
Roberts, J. D., Green, C., J . Am.
Chem. SOC.68.214 (1946 1. (14) Robinson, R., A-utwe 173,541 (1954). (15) Strain, H. H., J . -4m. Chem. SOC. 57,758 (1935). (16) White, J. IT.,ASAL. CHEM. 20, 726 (1948).
RECEIVEDfor review June 29, 1956. Accepted June 6, 1957. Scientific Article A-567. Contribution 2725 of the Masyland Agricultural Experiment Station, Dairy Department.
Interferences with Biuret Methods for Serum Proteins Use of Benedict's Qualitative Glucose Reagent as a Biuret Reagent RICHARD
J. HENRY, CHARLES SOBEL, and SAM BERKMAN
Bio-Science laboratories, 10s Angeles 64, Calif.
b The biuret determination of serum proteins was studied employing Benedict's qualitative glucose copper reagent. The ratio of nitrogen to color produced was the same for human albumin and human y-globulin. The biuret method also agreed within experimental error with the Kjeldahl analysis in a series of sera in which the paper electrophoretic patterns varied widely. Various proposed corrections for turbidity were studied, with the conclusion that the most universally successful correction was ether extraction. Bilirubin did not interfere as long as turbidity was not present. Hemolysis interfered, but when the hemoglobin concentration was independently determined, a correction could b e made. interferences produced b y ammonium ion and high salt concentrations varied with the particular biuret technique employed.
K
(IO) simplified the biuret procedure for determination of serum proteins by using a reagent relatively low in copper and high in alkalinity, so that the reaction could be carried out directly on serum without precipitation of cupric hydroxide. The high alkalinity kept the excess copper in solution. It has been claimed that this reagent is unstable because of its high alkalinity and several modifications haye been proposed, all aiming a t stabilization of the reagent a t low concentrations of alkali without the formation of insoluble cupric hydroxide. Thus, Nehl (16) used ethylene glycol as a complexing agent, and Weichselbaum (23) used sodium potassium tartrate for complexing and potassium iodide to prevent autoreduction. Gornall ( 5 ) retained the tartrate but believed it safe to omit the potassium iodide. Goa (3) has proposed, and the present authors INGSLEY
have also used for approximately 15 years, Benedict's qualitative glucme reagent as a biuret reagent. The citrate present complexes the excess copper in the reaction and the reagent is stable a t room temperature because of its low alkalinity. There are a t least four possible sources of interference with the biuret determination : 1. Turbidity. Four solutions to this problem have been proposed-namely, extraction with ether ( 8 ) , subtraction of a serum blank (18), preliminary precipitation of protein by trichloroacetic acid (ZO), and subtraction of residual absorbance after the biuret color has been dispelled by cyanide (7'). 2. Presence of Salts. Salts used in protein fractionation, such as sodium sulfate and sodium sulfite, have been reported to increase the color intensity (22). Ammonium ion has been observed to affect results but the error is VOL. 29,
NO. 10, OCTOBER 1957
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