Spectrophotometric Determination of Urea, Thiourea, and Certain of Their Substitution Products with p-Dimethylaminobenzaldehyde and Diacetylmonoxime R. C.
HOSENEY and K.. F. FINNEY
Hard Winter Wheat Quality Laboratory, Crops Research Division, ARS, USDA, Kansas State University, Manhattan, Kan.
b A method using p-dimethylaminobenzaldehyde for the determination of urea has been modifield and extended to determine urea, thiourea, and several substitution pr.oducts with increased sensitivity involving concentrations that were generally below 10 p.p.m. for the ureas and below 50 p.p.m. for the thioureas. Ureas reacting with p-dimethylaminobenzaldehyde gave a yellowish-green color with a broad transmittancy minimum from 425 to 445 mw. The thioureas gave the same color but transmittance minimums were 440 k 460 mp. The improved method substitutes isopropyl alcohol for water and sulfuric acid for hydrochloric acid. Using isopropyl alcohol as a solvent demands that extreme care be taken to exclude water from the reaction. As little as o.25Y0 water in the color solution decreased b y 50% the color produced with urea. Methods are given for the determination of five ureas (urea, methylurea, t-butylurea, allylurea, and phenylurea] and four thioureas (thiourea, allylthiourea, phenylthiourea, and eihylenethiourea). Determination of ethylenethiourea required a mixture of isopropyl alcohol and acetic acid as a solvent. To determine certain ureas not reacting with p-dimethylaminobenzaldehyde, a method using diacetylmonoxime for the determination of urea has been modified and extended to determine urea, thiourea, acetylurea, acetylthiourea, 1,3-dimethylurea, and 1,3-diethylthiourea. Altering the concentration of reagents increased color stability and extended the linear portion o f the absorbance vs. concentration curves over a greater’portion of the absorbance scale without decreasing sensitivity of the method.
A
to skudying glutenprotein quality (5, 6) is based on the ability of the wheat plant to take up, translocate, and synthesize into gluten protein the urea nitrogen sprayed on the leaves ( 7 ) . Subnormal physical and baking properties of some wheats compared with normal and abovenormal properties of others sprayed N E W APPROACH
with ureas suggested that certain ureas had been synthesized to varying degrees into gluten protein. Thus, a sensitive quantitative niethod is needed to determine residual quantities, if any, of the ureas and thioureas in wheat flour. Residual amounts of certain ureas and thioureas might account for the below- and above-normal properties of certain wheat flours. This paper describes modifications necessary to determine low concentrations of certain ureas and thioureas by spectrophotometric methods that employed p-dimethylaminobenzaldehyde and diacetylmonoxime. The classical method for determining urea consists of hydrolyzation with the enzyme urease, followed by the determination of ammonia by Xesslerization or the Kjeldahl procedure. This method is inappropriate for the present study because urease attacks only urea. TROadditional methods have been used to determine urea spectrophotometrically. One uses an acidic solution of p-dimethylaminobenzaldehyde. The Eecond method uses diacetyl and related compounds. Tt7att and Crisp ( I S ) described a spectrophotometric method that used p-dimethylaminobenzaldehyde and an optimum urea concentration range of 50 to 240 p.p.m. Several workers ( 2 >3, 8 ) used their method successfully without major modifications. I n earlier work, Barrenscheen (1) first described a color reaction of urea with Ehrlich’s aldehyde reagent, a solution of p-dimethylaminobenzaldehyde in hydrochloric acid. He reported that the reaction was equimolar and that the bright yellowish-green product, formed by the direct addition of the acid to form a salt, was relatively unstable. Increasing the hydrochloric acid concentration led to complete loss of color. The compound formed with sulfuric acid has greater stability. L-rea, methylurea, and phenylurea gave the same color. Systematic investigation of other ureas showed that an unbound amino group was essential for the react ion. Fearon (4)reported that color was produced by heating urea with diacetyl-
monoxime in strong hydrochloric acid solution. He found color intensity improved when a drop of 1% potassium persulfate was added. After a rather thorough study, he concluded that the reaction was positive with compounds containing the system RNHCONHR’, where R was either hydrogen or a simple aliphatic radical and R’ was not an acyl radical. Natelson, Scott, and Beffa (11) found that potassium persulfate enhanced the color intensity 10 to 15%, but also caused a gradual fading even in the absence of light. They showed that persulfate destroyed the hydroxylamine formed by hydrolysis of the monoxime and that hydroxylamine decreased the color produced by the reaction of diacetyl and urea. They described a method of using diacetyl instead of diacetylmonoxime. Diacetyl produced the same color as diacetylmonoxime, had a shorter reaction time, and did not require an oxidizing agent. This method was modified by LeMar and Bootzin ( I O ) to increase the reagent stability. h rather thorough study of the reaction of urea with diacetylmonoxime was undertaken by Rosenthal ( l a ) , who worked on Kawerau’s (9) modification of using arsenic acid to destroy the hydroxylamine as it was formed. EXPERIMENTAL
Apparatus. Spectrophotometric readings were taken on a Beckman DU spectrophotometer equipped with an a x . power supply, a photomultiplier tube, and a thermoregulated water bath. A matched set of Corex cells with a path length of 0.999 cm. was used. Readings were taken a t 25’ C . and a t a slit width of 0.01 mm. Materials. Practical grade ethylenethiourea (2-imidazolidmethione) was recrystallized twice from water. Practical grade t-butylurea a n d 1,3dimethylurea was recrystallized twice from 95y0 ethyl alcohol. All other ureas and chemicals were reagent grade. The ureas were dried overnight a t 70’ C. in a vacuum oven. p-DIMETHYLA-MI Pi O B E N Z A L D E H Y D E .
Pipet a n appropriate aliquot of the isopropyl alcohol extract containing urea VOL. 36, NO. 11, OCTOBER 1964
2145
methylaminobenzaldehyde. Because ethyl alcohol is more acidic and a better solvent for p-dimethylaminobenzaldehyde than water, samples were dissolved and diluted to volume with %Yo ethyl alcohol. Because hydrochloric acid contains a significant amount of water, sulfuric acid was tried and found to be equal to or better than hydrochloric acid. Changing the solvent to 95Y0 ethyl alcohol increased sensitivity (change in transmittance per unit of concentration). In addition, a color reaction was obtained in thiourea. The success of 9570 ethyl alcohol as a solvent warranted investigating a number of other solvents including absolute ethyl alcohol, n-propyl alcohol, isopropyl alcohol, sec-butyl alcohol, isobutyl alcohol, n-butyl alcohol, tertbutyl alcohol, and acetic acid. Absolute alcohol and n-propyl alcohol offered little or no advantage over 9570 ethyl alcohol. Difficulty was encountered in obtaining color with the butanols. Color intensity when using isopropyl alcohol or acetic acid as a solvent was several times that of 95% ethyl alcohol. The reaction with an acetic acid solvent was unstable. Therefore, with one exception, isopropyl alcohol was adopted as a solvent. Extreme care must be taken to exclude water from the reaction, because as RESULTS little as O.25y0 water in the color solup-DIMETHYLAMINOBENZALDEHYD E. tion decreased by 50% the color produced with urea. Thus, all samples CHOICEO F SOLVENT.With the method must be dried and the flasks or pipets of W a t t and Crisp ( I S ) , several of the either dried or rinsed with isopropyl monosubstituted ureas gave a color alcohol before being used. similar to that given by urea. ThioCOLORSOLUTIONS. Three color soluureas gave no color in water solution. tions were used to obtain maximum The p-dimethylaminobenzaldehyde sensitivity for each of the ureas and was only slightly soluble in water but thioureas. Color solution A consisted very soluble in strong acid. An excess of 2 grams of p-dimethylaminobenzof strong acid, however, depressed the aldehyde, 100 ml. of isopropyl alcohol, color reaction between urea and p-di-
or thiourea into a 25-ml. volumetric flask, add 10 or 15 ml. of the appropriate color solution, dilute to volume with isopropyl alcohol, and place in a water bath at 25' C. for 15 minutes. Color produced by the reaction of p-dimethylaminobenzaldehyde with ureas and thioureas, in general, is stable for a t least 1 hour. Photolabile color produced with thiourea and allylthiourea faded appreciably within 30 minutes, but was stable for over 1 hour with low actinic glass volumetric flasks. DIACETYLMONOXIME. Diacetylmonoxime, 2.5 grams, was dissolved in 100 ml. of 5y0 acetic acid. Concentrations of hydrochloric acid, arsenic acid, and diacetylmonoxime and heating time varied with the compound being investigated. Optimum conditions are specified in Table I. I n general, 10 ml. of acid mixture were added to appropriate urea extracts in 25 X 200mm. reaction tubes, followed by 1.5 or 2 ml. of diacetylmonoxime solution and sufficient water to adjust the volume to about 22 ml. Thereafter, the tubes were immersed in a rapidly boiling water bath. After being heated, the tubes were cooled in running water for 10 minutes, and their contents transferred to 25-ml. volumetric flasks and diluted to volume with distilled water.
Table I.
Optimum Conditions for Reaction of Certain Ureas and Thioureas with Diacetylmonoxime
Compound
Heating time, minutes
TTren
AZylurea 1,3-Dimethylurea Thiourea Acetylthiourea 1,3-Diethylthiourea
Table II.
mp 4x0 -_.
4n -_
50
485 550 480 485 510
20 45 35 30
Std. dev., p.p.m.
n io
0 0 0 0 14
24 75 43 48 18
Acid Mixture per 100 m1.HC1, AsnOs, grams ml. 60 0 60 0 60 0
60 0 70 0 60 0
0 7354 0 7354
0 7354
2 9410 0 7354 1 1030
Dia&,ylmonoxime solution, ml. 2 2 1 2 2
0 0 5 0 0
20
Summary of Optimum Conditions for Reaction of Certain Ureas and Thioureas with p-Dimethylaminobenzaldehyde
Compound Urea Methylurea t-Butylurea Allvlurea Phenylurea Thiourea Phenylthiourea Allylthiourea Ethylenethiourea
2 146
Wavelength,
Color solution
ANALYTICAL CHEMISTRY
A A A A A
B B A
C
Wavelength, mr 425 430 430 43 5 440 450 445 445 450
Concentration range, p.p.m.
Std. dev., p.p.m.
1-8 0.5-3.5 0.5-2.5 0.5-4 0 5-4 5-25 5-30 5-20 10-40
0.16 0.01 0.01 0.12 0 14 0 44 0 72 0 32 1 21
and 0.5 ml. of concentrated sulfuric acid. Each determination required 10 ml. Color solution B consisted of 2 grams of p-dimet hylaminobenzaldehyde, 100 ml. of isopropyl alcohol, and 1 ml. of sulfuric acid. Each determination required 10 ml. Color solution C consisted of 2 grams of p-dimethylaminobenzaldehyde, 150 ml. of glacial acetic acid, and 0.6 ml. of sulfuric acid. Each determination required 15 ml. Fresh color solutions must be prepared each day. The color solution, wavelength, and concentration range for each compound are specified in Table 11. The standard deviations were determined over the concentration range and are for a single determination (30 replications). Transmittancy curves showing the broad transmittancy minimums for each compound are given in Figure 1. Ethylenethiourea presented a special case. The color intensity produced in isopropyl alcohol solvent was low and that produced in acetic acid solvent was unstable. A combination of the two solvents gave good color intensity and stability. Thus ethylenethiourea dissolved in isopropyl alcohol was added to 15 ml. of acetic acid color solution C. Dilution to 25 ml. was made with isopropyl alcohol. ~ ~ P P L I C A T I O NFlour . samples, to which known amounts of a urea or thiourea had been added, were dried overnight a t 70' C. in a vacuum oven. X 2-gram flour sample containing 12.5 mg. of a urea or thiourea was agitated in a reciprocating shaker for one hour with 100 ml. of isopropyl alcohol. After filtering through Whatman S o . 4 filter paper, analyses were made on aliquots of the filtrate. Data for the analyses are given in Table 111. Six of the nine ureas gave recoveries of slightly over 100%. DIACETYLMONOXIME. Spectrophotometric readings were taken a t the wavelengths shown in Table I. Absorption curves for each of the compounds are shown in Figure 2. Best results were obtained when readings were taken within one hour. Standard deviations given in Table I are for a single determination (30 replications). Although no fading by light was encountered in this study, the tubes were heated under subdued light. Choice of Reagent. It appeared from the literature t h a t diacetyl as a reagent (11) had the advantages of requiring no oxidizing agent and less heating time than diacetylmonoxime and would produce the same color. In preliminary experiments, diacetyl gave the color and absorption curve, but only about one tenth the color intensity of diacetylmonoxime. No conditions were found where diacetyl gave a color of the same intensity as that with diacetylmonoxime. The more intense color with diacetylmonoxime can be attributed to slow production of diacetyl and continuous destruction of the generated hydroxylamine.
"1 IO
UREA
I
THIOUREA
-.-u 420
?
ALLYLURE A
440
425
445
PHENYLUREA
9 0 \ METHYLUREA
440
465
1
465
490
500
470
I
\.-.-.-.-.
IO
L
425
450
440
.
.-.-/./*
IO
I
15
a_._._.-.
ALLYLTHI~UREA
-.
*A
*\
*\:-*-*-*,e
15
.-.-.-./e
10
*-.-.-._.A'
10
.-.-._.-a
. 470
500
5
5
50.
.-.-.-.., . *.._._._./. -.-./-
460
..._.-.-. .-._._.-. .-.-._._.
90. t-BUTYLUREA
30
-
ETHYLENE THIOUREA
I , 3 - 0 IETHYLUREA
440
520
.
4 95 50
/*
loo 150
I,3-DIETHYLTHIOUREA
u 420
440
435
455
WAVE LENGTH
Figure 1.
460
- my
Transmittance vs. wavelength for reaction of
p-dimethylaminobenzaldehyde with each of nine ureas or thioureas a t each of three concentrations in parts per million
Table 111. Analyses of Flours Containing Knbwn Quantities of Certain Ureas or Thioureas with p-Dimethylaminobenzaldehyde
Average QuanQuantity tity ~ e ComAdded, Found, found, covery, pound mg. mg. mg. 70 Urea 1 2 . 5 12.74 12.67 101 4 12.5 12.74 12.5 12.53 Methvl- 1 2 . 5 12.89 12 77 102 2 12 89 12,54 &Butyl- 1 2 . 5 12 11 12 02 96 4 urea 1 2 . 5 12 04 1 2 . 5 11.90 Allylurea 12.5 13 31 13 29 106 3 1 2 . 5 13 58 12.5 12 98 Phenyl12 5 12 46 12 55 100 4 urea 12 5 12 48 12 5 12 72 Thiourea 12 5 13.13 13 32 106 5 12 5 13 48 12 5 13 35 Phentl- 12 5 12 39 12 31 98 5 thiourea 12 5 12 22 12 5 12 32 Allyl12 5 12 17 12 12 97 0 thiourea 12 5 12 18 12 5 12 02 Ethylene- 12 5 12 13 12 80 102 2 thiourea 12 5 13 49 12 5 12 77
580
480
530
WAVE LENGTH - r n ~
Figure 2. Transmittance vs. wavelength for reaction of diacetylmonoxime with each of six ureas or thioureas at each of three concentrations in parts per million
Application. -4nalyses were made after adding a known amount of urea or thiourea to a n aliquot of a flour composite. A 2-gram sample of flour containing the urea or thiourea being investigated was agitated in a reciprocating shaker for 1 hour with 100 ml. of water; it was then filtered through Whatman KO. 4 filter paper. Analyses for specific ureas were performed on aliquots of the filtrates. Data from the analyses are given in Table IV. Five of the six ureas gave recoveries of slightly less than lOOyo. Trea and thiourea, the only two compounds used with both reagents, have high recoveries with p-dimethylaminobenzaldehyde and low recoveries with diacetylmonoxime, the average of the two methods being very near 1 0 0 ~ o . DISCUSSION
p - Dimethylaminobenzaldehyde. T h e modified method for urea decreases the detectable concentration from 50 to 1 p.1i.m. and increases the sensitivity about 25 times. T h e method was extended to determine eight other compounds. The broad and overlapping absorption peaks make it impossible to determine a n y one urea or thiourea in the presence of one or more of the others. Barrenscheen (1) stated that a urea or thiourea must have a free amino group to react. The prebent study included six compounds (1,3-dimethylurea; 1,3diethylthiourea, 1,3-diphenylurea; 1,3-
diphenylthiourea; 1,3 - diethyl - 1,3diphenylurea; and ethylenethiourea) that did not have a free amino group; ethylenethiourea was the only one that reacted contrary to the rule. Four compounds possessing a free amino group (1,l - diphenylurea; 1 , l - diphenylthiourea; acetylurea; and acetylthiourea) did not give the color reaction.
Table IV. Analyses of Flours Containing Known Quantities of Certain Ureas or Thioureas with Diacetylrnonoxime
Average Quan-
-
Compound Urea Acetylurea 1,3-Dimethylurea Thiourea Acetylthiourea 1,3-T>iethylthiourea
~ Quantity_
tity
R _
~-
Added, Found, found, covery, mg. mg. mg. % 12 5 12 06 12 22 97 8 12 5 12 35 12 5 12 25 12 5 12 04 12 34 98 7 12 5 12 40 12 5 12 58 12 5 12 45 12 36 98 9 12 5 12 11 12 5 12 53 12 5 12 22 11 85 94 8 12 5 11 57 12 5 11 57 12 5 12 79 12 62 100 9 12 5 12 17 12 5 12 89 25 0 26 8 24 5 98 0 25 0 23 6 25 0 24 0
VOL. 36, NO. 1 1 , OCTOBER 1964
2147
color was noted with 1,1diethylurea, but no method was established because a sample of eufficient purity was not obtained. The solution of p-dimethylaminobenzaldehyde in isopropyl alcohol (color solution A) was an extremely sensitive spray reagent for detection of certain ureas and thioureas on paper chromatograms. Brilliant yellow spots on a white background were obtained a t room temperature. The urea in perspiration will give a spot if the papers are touched with bare hands. Although no attempt was made to apply the reaction to samples other than wheat flour, other materials including feeds containing added ureas should work equally well, particularly if they can be dried and extracted with isopropyl alcohol. If feed pigments interfere, Jongen and Berkhaut’s (8) method may be used to clarify and decolorize the sample in one step. Diacetylmonoxime. T h e reaction of diacetylmonoxime with urea is not understood. Natelson, Scott, and Beffa (11) found the active reagent to be diacetyl, not diacetylmonoxime. Diacetylmonoxime produced a greater color intensity than diacetyl in the present study. Appreciable amounts of
the volatile diacetyl probably are lost before reacting. X slow production of diacetyl by the hydrolysis of the oxime probably permits a more favorable reaction. Fearon (4) stated that the reaction was positive for compounds containing the system RKHCONHR’, where R is hydrogen or a simple aliphatic radical and R‘is not an acyl radical. Contrary to Fearon’s statement, a positive reaction was obtained with acetylurea and acetylthiourea in the present study. No color was produced with 1,3diphenylurea; 1,l-diphenylurea; ethylenethiourea; 1,3-diphenylthiourea, or 1,l-diphenylthiourea. A positive reaction was obtained with 1,l-diethylurea but no method was established because of the difficulty in obtaining a sample of sufficient purity. The reaction with diacetylmonoxime apparently is positive for compounds containing the system RNHCO(S)XR’R”,where R is hydrogen or a simple aliphatic radical; R’ is hydrogen, a simple aliphatic radical, or a phenyl group; and R ” is less complex than a phenyl group. Fearon (4) reported that diacetylmonoxime reacts with protein, but in the present study no reaction was noted with the water-soluble extracts of flour.
LITERATURE CITED
( 1 ) Barrenscheen, H. K., Biochem. 2. 140,426-34 (1923). ( 2 ) Brown, H. H., ANAL. CHEM.31, 1844-6 il9.W) \ - - - - /
( 3 ) Cline, R. E., Fink, R. M., Zbid., 28, 47-52 (1966). 14) Fearon, W. R., Biochem. J . 33, 902-7 (1939). ( 5 ) Finney, K. F., Trans. Am. Assoc. Cereal Chemists 12, 127-42 (1954). ( 6 ) Finney, K. F., unnumbered mimeo-
graph publication, presented at Fertilizer Conference K.S.U., Manhattan, Kan., December 1950. ( 7 ) Finney, K. F., Meyer, J. W., Smith, F. W,, Fryer, H. C., Agron. J . 49, 341-7
(1957). (8) Jongen, G. H., Berkhaut, H. W., Chern. Weekblad 52, 909-10 (1956): C.A. 51, 5635 (1957). ( 9 ) Kawerau, E., Sei. Proc. Roy. Dublin SOC. 42, 63-70 (1946); C.A. 40, ,5085 (1946). (10) LehIar, R. L., Bootzin, David, ANAL. CHEM.29, 1233-4 (1957). (11) Natelson, Samuel, Scott’, M. L. Beffa, Charles, Am. J . Clin. Pathol. 21, 275-81 (1951). (12) Rosenthsl, H. L., ANAL.CHEM.27, 1980-2 (19%). (13) Watt, G. W., Crisp, J. D., Ibid., 26, 452-53 (1954). Received for review April 7, 1964. Accepted July 24, 1964. Cooperative in-
vestigation between the Crops Research Division, ARS, LSDA, and the Department of Flour and Feed Milling Industries, Kansas State University, Manhattan Kan. Contribution No. 437, Department of Flour and Feed Milling Industries.
Analysis for Deuterium in Organic Compounds by Combustion-infrared Spectrometry JAMES L. LAMBERT, JAMES H. HAMMONS, JOSEPH A. WALTER, and ALEX NICKON Department o f Chemistry, The Johns Hopkins University, Baltimore, Md. 2 7 2 7 8
b A method is described for the quantitative analysis of deuterium in compounds by combustion and infrared spectrometry of the derived water sample, Combustion is effected conveniently by a dynamic combustion train, modified to remove acidic oxides and halogen from the combustion gases. The memory effect of this system has been reduced to an acceptable level, Analysis on the milligram scale is possible. Results agree well with analyses by mass spectrometry and the falling-drop method.
T
AND CONDON first described an infrared method for the determination of deuterium oxide in water (8). This method depends on the fact that HOD has an absorption band a t 2520 cm.-’ (3.97 microns) that can be observed through the overlapping spectrum of HOH. The intensity of the HOD band provides a direct measure of the total deuterium content of the water
HORNTON
2 148 *
ANALYTICAL CHEMISTRY
sample a t high isotopic dilution, where the concentration of DOD is negligible. Trenner, Arison, and Walker developed this technique for the determination of deuterium in organic compounds by assay of the water formed on combustion. In their early work (10) these investigators used the conventional Pregle microanalytical type of combustion train for the oxidation of the organic compound. They found the usefulness of this dynamic combustion technique limited by a serious memory effect attributed mainly to the barium carbonate used to remove acidic oxides. Subsequently they developed a static oxidation method in which the organic compound was heated at 750’ to 800’ with a large excess of copper oxide in a sealed quartz tube (9). The water sample was then recovered and purified in a 7-acuum distillation train. Jones and MacKenzie ( 1 ) further refined the method and suggested a number of procedural modifications regarding the methods of sample handling and of
water collection. These latter workers overcame the difficulties associated with temperature changes on the spectrometry of deuterium-enriched water by employing differential analysis against natural-abundance water. The static combustion method entails considerable inconvenience and the disadvantages concomitant with complex techniques. The quartz combustion ampule must be carefully prepared and heated to avoid explosive loss; and to recover and purify the water sample obtained from combustion, a complex ampule-breaker/distillation apparatus is required together with careful manipulation. We have simplified the method by returning to the dynamic combustion train technique for the combustion of the deuterium-enriched compound. In our method water obtained from combustion of compounds containing nitrogen, sulfur, and halogen was purified in the combustion train by use of suitable absorbents which removed acidic nitrogen, sulfur oxides, and halo-
