ISSN 0027�1314, Moscow University Chemistry Bulletin, 2011, Vol. 66, No. 6, pp. 345–350. © Allerton Press, Inc., 2011.Original Russian Text © E.M. Basova, M.A. Bulanova, V.M. Ivanov, 2011, published in Vestnik Moskovskogo Universiteta. Khimiya, 2011, No. 6, pp. 419–425.

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The indicator method of investigation for interwellspace of an oil deposit is widely used in geophysics forthe control of displacement of oil by flood water [1].The method is based on the injection of the requiredvolume of liquid labeled with the indicator into thestudied bench through the injection well, edging it bythe flood water to controllable extraction wells, andinvestigation of time change of indicator concentra�tion in liquid flow outward from the bench. The resultsof the indicator study are used to create geological andhydrodynamic models of oil fields, for estimation ofoil reserves, and projecting for mining. Whereas, whenmoving the indicator over the reservoir, dilution up to104 times takes place, it is necessary to have a methodof control of its content at the level of 1 mg/L.

As indicators, fluorescent dyes, phosphate�, thio�cyanate�, and nitrate�ions are widely used [1]. Ureameets all the requirements of the indicators in geo�physical studies [1]: very soluble in water, doesn’taffect the processes of oil refining, environmentallysafe, cheap, and provides cost efficiency of indicatorstudies. Urea is used as an indicator in the Republic ofBelarus. Simultaneously, another 3–4 indicators areused, which request methods of selective determina�tion for each of them and there is no disturbing influ�ence of other indicators.

There are a few methods of urea determination.Total nitrogen content in end products is controlled bythe titrimetric method after decomposition of urea byconcentrated H2SO4 and converting it into ammo�nium ion [2]. The sample mass is 1 g in the determina�

tion of by the formaldehyde method and 5 gwhen using the predistillation of ammonia. The titri�metric method of analysis is not suitable for determi�nation of a low content of urea.

NH4+

The determination of urea in blood, urine, andother biological fluids is based on the quantitativetransfer of it into ammonium carbonate by the enzymeurease with subsequent spectrophotometric determi�

nation of ions by the Nessler reagent [2, 3]. Themethod allows us to determine 5–20 g/L of urea nitro�gen [3]. Organic nitrogen in natural and waste watersis also determined by the Nessler reagent after decom�position by the concentrated H2SO4 [3, 4].

While analyzing natural waters a voluntary volumeof the sample should be 500 mL, in addition, ammoniais predistilled from the alkaline solution [3]. Whileanalyzing waste waters 100 mL of the sample isenough, and the determination is carried out directly(without distillation) [3]. Spectrophotometric deter�mination of ammonium ions by the Nessler reagent issensitive enough—from 0.05 to 4.00 mg/L in naturaland waste waters, 50 mL of the sample is required foranalysis [5]. Whereas the samples of natural waters, aswell as biological fluids, already contain ammoniumnitrogen, its concentration should be established pre�viously and then subtracted from the concentration,and obtained after the decomposition of urea. The dis�advantage of the method is the necessity to work withnon�ammonia water (as one way bidistilled water ispassed through a column with the cation exchangerKU�2).

Direct spectrophometric determination of urea isof great interest. The appearance of yellow�greencoloration in the process of reaction of the deter�mined solution with p�dimethylaminobenzaldehyde(DMABA) in the presence of HCl is used for thedetection of urea, and the detection limit is 2 mg/L[6]. The method of spectrophometric determinationof urea in the reservoir water using DMABA is laid

NH4+

Photometric Detection of Urea in Natural WatersE. M. Basovab, M. A. Bulanovab, and V. M. Ivanova

aDivision of Analytical Chemistry, Department of Chemistry, Moscow State University, Moscow, Russiae�mail: mvonavi@mail.ru

bDubna International University of Nature, Society, and HumansReceived February 1, 2011

Abstract—The photometric detection of urea with the use of p�dimethylaminobenzaldehyde as a reagent hasbeen develeped. The method allows one to reliably determine 10 mg/L of urea with the volume of an aliquotof 10 mL. The method has been applied for the determination of urea in river water.

Keywords: urea, p�dimethylaminobenzaldehyde, natural water, photometric detection.

DOI: 10.3103/S0027131411060022

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out on the internet in the analytical forumANCHEM.RU and characterized by a minimumdetectable concentration (2 mg/L) [7]. Formation ofthe yellow compound of urea with DMABA underliesthe method of the determination for mass fraction ofamide nitrogen in fertilizers. The range of detectablecontents is 20–46 mass % [8]. Thereby, to construct acalibration curve, 160–320 mg of urea were added into100�mL flasks. In summary, the method [8] allows oneto analyze water samples with the concentration of1.8–3.6 g/L, which is three orders of magnitudegreater than in method [7].

The purpose of this work is to estimate the reliabil�ity of information from the internet and developmentof the method for detection of urea in natural waters bythe spectrophotometric method using DMABA.

EXPERIMENTAL

Reagents. Pure urea (EKROS, Russia), analyti�cally pure DMABA (Laboratornaya tekhnika, Rus�sia), CH3COOH, 99.5% (Panreac, Spain), and ana�lytically pure conc. HCl (EKROS, Russia) were used.Distilled water was also used in the work.

Solutions. Standard (2.5 g/L) solution of urea wasprepared by dissolving an exact weight of specimen inwater. The process solution with the concentration ofurea 250 µg/mL was prepared by diluting the initialstandard solution with water.

To prepare the solution of DMABA 20 (or 40) g ofreagent was dissolved in 50 (or 100) ml of conc. HCl ina 1�L volumetric flask, diluted with water to the markand stirred. The next day the solution was filtered. Adiluted solution of hydrochloric acid was prepared bydiluting 5 mL concentrated solution in a 100�mL vol�umetric flask with water.

Apparatus. The absorbency of solutions was mea�sured on photometers KFK�2MP or KFK�3 at 400 or440 nm, respectively, in cells with l = 5 cm relative tothe solution of the control experiment.

Experimental technique. The solutions of urea,DMABA, acetic acid, and HCl (if necessary) wereadded sequentially into 25�mL volumetric flasks,diluted with water to the mark and stirred. The absor�bency was measured relative to the reference solution.

RESULTS AND DISCUSSION

Reproduction of the Technique from the internet[7]. The solutions contained 15 mL of water, 10 mL ofreagent DMABA with a concentration of 20 g/L, and5 mL of CH3COOH; the total volume of solutions was35 mL. Dependence of absorbency on urea concen�tration in water is shown in Table 1. It is linear,described by the equation A = 0.00324c with a correla�tion coefficient of r = 0.998, but values of absorbencyare very low. Therefore, minimum detectable concen�tration of urea can not be 2 mg/L, as indicated in work[7]. Objectively, surely water samples with a concen�tration of urea over 15 mg/L can be analyzed.

Selection of optimum conditions for the complex�ation of urea with DMABA. A compound of urea withDMABA has a maximum absorption at 420 nm [8]. Itwas shown that the absorption of solution on KFK�2MPis maximum when using a color�filter with transmissionwavelength of 400 nm, and on KFK�3—440 nm.

Influence of DMABA, HCl, and CH3COOH con�centration on the absorbency was studied, and theresults are presented in Table 2. It can be seen that theintroduction of CH3COOH increases the absorbencyby 1.5 times. It should be noted that in technique [8]this acid was not added to the solution. The optimumcontent of conc. CH3COOH is 5 mL. Additional add�ing of HCl is inappropriate.

Dependence of the absorbency on the concentra�tion of DMABA is complicated: one can clearly dis�tinguish two plateaus in the range of 6–8 mL and 12–18 mL for the solution of DMABA with a concentra�tion of 20 g/L. Thereby, introduction of a largeamount of reagent leads to an increase in absorbencyof the solution that should allow us to detect lowerconcentrations of urea. As the best, 7 and 12 mL ofDMABA solutions (20 g/L) were chosen. Since theintroduction of a large volume of reagent solutiondecreases the volume of aqueous solution containingurea the solution of DMABA with a concentration of40 g/L was prepared whose optimum volume is 6 mL.

The change of the absorbency for the solution ofthe complex on standing was studied. It was shownthat the absorbency of the solution is constant for5 min after mixing the solutions for both concentra�tions of reagents, then, decreases slightly. So, in thesolution containing 7 mL of DMABA (20 g/L), theabsorbency decreased by 11% after 45 min. In thesolution containing 6 mL of DMABA (40 g/L), theabsorbency decreased by 6% after 15 min, and by 8%after 25 min. So, the absorbency of solutions should bemeasured for 5 min after preparation.

Complex formation reaction of urea with DMABA.Stoichiometry of the components in the process ofcomplex formation was studied by molar�ratiomethod [9]. For this a dependence of the absorbencyof solutions with constant concentration of DMABA,

Table 1. Dependence of the absorbency on the concentra�tion of urea in water

c, mg/L 1 5 10 15 20 25

A (400 nm) 0.005 0.016 0.033 0.046 0.068 0.080

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PHOTOMETRIC DETECTION OF UREA IN NATURAL WATERS 347

equal to 0.01072 M on the concentration, was con�structed (Table 3).

It can be seen, that the break point on the satura�tion curve is unclear, the molar ratio of DMABA andurea, obtained by extrapolation of linear segments ofthe curve to intercrossing, is 2.08 : 1.0, which is close

to 2 : 1. An unclear kink on the saturation curve indi�cates the slight stability of the complex formed.

Urea has the properties of a nucleophile and weakbase [10]. Reactions of carbonyl compounds withbases are catalyzed by both strong and weak acids [11].Interaction between urea and DMABA can be repre�sented by the following scheme.

Since urea is a weak base (pKb = 13.82), pulling theelectrons of the carbonyl group with acid catalyst is

necessary to increase the reactivity of the carbonylgroup towards nucleophile—nitrogen atom of urea.

O CNH2

NH2

C

N(CH3)2

O

H

C

N(CH3)2

O

H

O C

NH

NH

C(OH)

C(OH)

N(CH3)2

N(CH3)2

+

+

Scheme.

O CNH

NH2

C(OH) N(CH3)2

Table 2. The effect of quantity of reagents on the formation of colored compound of urea with DMABA (total volume ofsolution is 25 mL, 20 g/L of DMABA, l = 5 cm, 400 nm, KFK�2MP)

Urea added, mgAdded, mL

ADMABA CH3COOH HCl

1.250 2.00 7.00 0 0.0504.00 7.00 0 0.0426.00 7.00 0 0.1148.00 7.00 0 0.113

10.00 7.00 0 0.15012.00 7.00 0 0.16214.00 7.00 0 0.238

0.500 2.00 5.00 8.00 0.0154.00 5.00 6.00 0.0605.00 5.00 5.00 0.1006.00 5.00 4.00 0.1227.00 5.00 3.00 0.1228.00 5.00 2.00 0.127

10.00 5.00 0 0.09512.00 5.00 0 0.17516.00 5.00 0 0.17018.00 5.00 0 0.180

0.500 6.00 5.00 4.00 0.1226.00 5.00 6.00 0.1036.00 5.00 10.00 0.080

0.500 6.00 3.00 4.00 0.1146.00 5.00 4.00 0.1226.00 7.00 4.00 0.1126.00 10.00 4.00 0.093

1.250 14.00 0 0 0.15314.00 7.00 0 0.238

0.250 12.00 0 0 0.08012.00 5.00 0 0.120

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Therefore, the reaction should occur only in acidicmedium. However, in strongly acidic medium prefer�ential protonation of a nucleophilic agent (urea is pro�tonated at the oxygen atom rather than nitrogen [10])and reduction of the reactivity of a free electron pairtake place, which leads to reduction of the degree ofcomplexation—to a decrease in the absorbency withincreasing the concentration of HCl and CH3COOH(Table 2). It is not possible to significantly decrease theconcentration of HCl or replace it with acetic acid,because it is necessary to dissolve the reagent. In work[8] for the preparation of 1 L of DMABA solution(4 g/L) 40 mL of conc. HCl was used.

Despite the fact that the stoichiometry of the form�ing complex of urea with DMABA corresponds to theratio of 1 : 2, excess reagent, required to reach a con�stant value of absorbency at fixed content of urea of500 µg is large—96� and 193�fold for the first and thesecond plateau, respectively (Table 2). Probably, in thearea of the first plateau complex 1 : 1 is dominated, andin the area of the second—1 : 2. Differences in the sta�bility constants of the complexes are negligible, sowhen constructing the saturation curve two points ofintersection were not obtained, but only one.

Metrological characteristics of the technique. Toconstruct calibration curves, the work solution of urea,7 mL of DMABA (20 g/L) or 6 mL of DMABA (40 g/L),and 5 mL of CH3COOH solutions are added into25�mL flasks, diluted with water to the mark, andmixed. For 5 min the absorbency of the solutions ismeasured at 400 nm (KFK�2MP) or 440 nm (KFK�3)in 5 cm cells relative to the solution of the controlexperiment. Calibration curves are linear in the rangeup to 750 µg of urea. The equations of calibrationcurves are presented in Table 4. When constructing thecurves an average of two measurements was used.While determining 375 µg of urea the relative standarddeviation was 0.06 (n = 6, 7.00 mL of reagent solutionwas added, the absorbency is 0.125–0.141), and whiledetermining 125 µg—0.12 (n = 6, 7 mL of reagentsolution was added, the absorbency is 0.030–0.039).

Average values of the molar absorption coefficientsfor the regions of the first and second plateau, whichdiffer by almost two times at 440 nm, are calculated(Table 4), which may also give evidence about the for�mation of the complex 1 : 2 at high concentrations ofreagents, because of the color of the compound causedby the presence of DMABA structure in the molecule.

Devices KFK�2MP and KFK�3 can determine theabsorbency in the range of 0.0–2.0. The value of

absorbency, which can be measured with the requiredaccuracy (sr < 0.33), is about 0.01 [12]. For use in thework cells with l = 5 cm and calculated values of themolar absorption coefficient (Table 4), detection lim�its are 30 µg (7 mL of DMABA solution with a con�centration of 20 g/L is added) or 20 µg (6 mL ofDMABA solution with a concentration of 40 g/L isadded). The recommended concentration range of urea is100–750 µg (in a 25�mL flask).

Analysis of the objects. The technique was used toanalyze a sample of river water by the method of“introduced�found.” A sample of river water wasselected on 11/23/2010 in the Uglich reservoir, Volgariver, Kalyazin city (FGVU “Tsentrregionvodhoz”Dubna ecoanalytical laboratory). The sample of watercontained calcium�, magnesium�, sulfate�, chloride�,nitrate�, and silicate�ions at the level of tens and unitsmg/L; pH was 7.39.

The sample of water had the highest chroma—46 degrees at MAC (maximum allowable concentration),for fishing ponds—20. Color was determined photo�metrically at the wavelength of 436 nm [13]. It wasshown that the absorbency for 10 mL of the sample,diluted with water to 25 mL, measured at 440 nm, is0.019 (an average of two measurements). Thus, in theprocess of analysis of natural samples the absorption ofthe matrix at the chosen wavelength should be takeninto account.

Performing the detection. The sample of analyzingwater is passed through the filter “blue ribbon.” Ten mLof the sample is put into 25�mL flasks, 6 mL ofDMABA with a concentration of 40 mg/L (or 7 mL ofDMABA with a concentration of 20 g/L), and 5 mL ofCH3COOH are added, diluted with water to the mark,mixed, and the absorbancy is measured in 5 min at 400or 440 nm in the cells with l = 5 cm. Optical density,which is determined by the color of initial sample, issubstracted from the optical density obtained. For thispurpose, 10 mL of the water analyzed is introduced in10�mL flask, 5 mL CH3COOH is added, diluted by waterto mark, stirred, and optical density is measured relativeto water at 400 and 440 nm in the cells with l = 5 cm.

Urea content in an aliquot of the sample (m, µg) isfound from the calibration curve. Concentration ofurea (c, mg/L) in the sample is calculated according tothe formula c = m/10. The results of detection are pre�sented in Table 5. The technique allows one to analyzereliably samples with a content of urea 10 mg/L andhigher.

Table 3. Dependence of the absorbency of solutions with a constant concentration of DMABA, equal to 0.01072 M,on the concentration of urea

curea, mg 1.25 2.50 5.00 7.50 10.00 12.50 15.00 20.00 25.00 30.00 37.50

A (440 nm) 0.102 0.230 0.400 0.530 0.616 0.694 0.723 0.737 0.750 0.760 0.766

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PHOTOMETRIC DETECTION OF UREA IN NATURAL WATERS 349

Concentrating. To reduce the lower limit of thedetected content the possibility of concentrating wasstudied. The easiest way to concentrate water samplesis evaporation. However, in boiling water urea hydro�lyzes with formation of ammonia and carbon dioxide,moreover, this reaction is catalyzed by acids and bases[10]. Evaporation of the model solution, prepared

with the use of distilled water, was studied. It was evap�orated to a volume of ~8 mL on an electric stove, care�fully watched, and left there to dry. As is seen from thedata of Table 5, error in the determination of urea inthe model solution after evaporation does not exceed11%. After evaporation of 100 mL of river water withthe addition of urea satisfactory results were also

Table 5. The results of urea detection in samples by the method of introduced�found (P = 0.95)

MatrixSample volume,

mL

c, mg/L

Found, µm n Found, µg c, mg/L sr D, % Conditions

River water

10 10.0 100 3 103 ± 7 10.3 ± 0.7 0.03 +3 KFK�3, 440 nm, 6 mL of DMABA (40 g/L)

10 20.0 200 3 191 ± 13 1.9 ± 1 0.03 –4.5

Distilled water

100 1.25 125 4 135 ± 16 1.4 ± 0.2 0.07 +8 KFK�3, 440 nm, 6 mL of DMABA (40 g/L), evaporating

100 3.75 375 3 336 ± 54 3.4 ± 0.5 0.06 –10.4

River water

100 5.0 500 3 467 ± 48 4.7 ± 0.5 0.04 –6.6 KFK�3, 440 nm, 7 mL of DMABA (20 g/L), evaporating

100 2.0 200 3 140 ± 66 1.4 ± 0.7 0.19 –30 KFK�3, 440 nm, 6 mL of DMABA (40 g/L), evaporating

Table 4. Calibration curves for the detection of urea

Apparatus (λ, nm) VDMABA, mL murea, μg A n ε, mol–1 l cm–1 Equation r

KFK�2MP (400) 7 (20) 125 0.042 6 78 A = 0.00024m 0.996

250 0.067

375 0.096

500 0.119

625 0.152

750 0.164

KFK�3 (440) 6 (40) 25 0.013 9 158 A = 0.00051m 0.973

50 0.029

100 0.050

150 0.078

200 0.110

250 0.135

375 0.192

500 0.260

625 0.305

KFK�3 (440) 7 (20) 125 0.041 6 97 A =0.000326m 0.998

250 0.081

375 0.123

500 0.162

625 0.204

750 0.245

350

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BASOVA et al.

obtained (Table 5). In the latter case, two more sam�ples were evaporated in parallel in order to take intoaccount the contribution of the color of the initialsample of water in the absorbency of the complex; theaverage value of the absorbency, due to the color, was0.111. It should be noted, that the pH of the analyzingsample of water was close to neutral. If the pH of thesample differs greatly from neutral, the sample shouldbe neutralized to reduce the catalytic effect of hydrox�onium� or hydroxide�ions.

Performance of the detection with preconcentrationby evaporation. A sample of analyzed water is passedthrough the filter “blue ribbon,” pH is measured,diluted solutions of HCl or NaOH are added dropwiseto reach a value of pH 6.5–7.5, if it is necessary. Onehundred mL of the sample is placed into a 100–150�mLbeaker, evaporated on a rangette to a volume of ~8 mL.The solution is quantitatively placed into a 25�mLflask, rinsing with 5 mL of water, 6 mL of DMABAsolution (40 mg/L), and 5 mL of CH3COOH areadded, diluted with water to the mark, and mixed.Within 5 min absorbency of solutions is measured at400 or 440 nm in cells (l = 5 cm). From the obtainedvalue of absorbency, absorbency, caused by the color ofinitial sample, is subtracted. For this, 100 mL of ana�lyzed water is evaporated on a hot plate to a volume of~8 mL, placed into 25�mL flask, 5 mL of CH3COOHis added, diluted with water to the mark, mixed, andabsorbency is measured at 400 or 440 nm in 5�cm cellsrelative to water. Urea content in an aliquot (m, µg) isfound by the calibration curve. Concentration of urea(c, mg/L) in the initial sample is calculated by theequation: c = m/100.

The results are presented in Table 5. In both cases,the found content of urea is less than the introducedcontent, which may indicate the partial hydrolysis ofanalyte; a degree of hydrolysis is higher in a moredilute solution. The technique allows one to analyzereliably samples with the urea content of 2 mg/L.

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