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A modified diacetyl
monoxime method for
colorimetric determination
1
of urea in soil extracts
a a
R. L. Mulvaney & J. M. Bremner
a
Department of Agronomy, Iowa State
University, Ames, Iowa, 50011
Version of record first published: 11 Nov 2008.

To cite this article: R. L. Mulvaney & J. M. Bremner (1979): A modified diacetyl

1
monoxime method for colorimetric determination of urea in soil extracts ,
Communications in Soil Science and Plant Analysis, 10:8, 1163-1170

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 COMMUN. IN SOIL SCIENCE AND PLANT ANALYSIS, 10(8), 1163-1170 (1979)

A MODIFIED DIACETYL MONOXIME METHOD FOR COLORIMETRIC DETERMINATION

OF UREA IN SOIL EXTRACTS 1

KEY WORDS: urea, soil extracts, diacetyl monoxime

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R. L. Mulvaney and J. M. Bremner

Department of Agronomy
Iowa State University
Ames, Iowa 50011

ABSTRACT

Some batches of phosphoric acid contain impurities that affect

colorimetric determination of urea in soil extracts by the method

of Douglas and Bremner, which involves measurement of the red

color formed when an aliquot of extract is heated with diacetyl

monoxime and thiosemicarbazide in the presence of phosphoric and

sulfuric acid. A modification of this colorimetric procedure is

described that permits use of phosphoric acid containing these

impurities and allows accurate and precise determination of urea

in soil extracts.

1163

Copyright © 1979 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part
may be reproduced or transmitted in any form or by any means, electronic or mechanical, including
photocopying, microfilming, and recording, or by any information storage and retrieval system,
without permission in writing from the publisher.
 1164 MULVANEY AND BREMNER

INTRODUCTION

The rapidly increasing importance of urea as a nitrogen

2-4
fertilizer in world agriculture has increased the need for

information concerning the fate of fertilizer nitrogen added to

soils as urea. To obtain such information, it is essential to

have a satisfactory method of determining urea in soils.

Douglas and Bremner showed that urea can be extracted quanti-

tatively from soils by 2M KC1 containing phenylmercuric acetate

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(a urease inhibitor) and that urea in such extracts can be

determined quantitatively by a colorimetric procedure involving

measurement of the red color formed when an aliquot is heated

with diacetyl monoxime and thiosemicarbazide in the presence of

phosphoric and sulfuric acid. This method has been used exten-

sively for determination of urea in research on urease activity

and urea transformations in soils and has been applied success-

fully to a wide range of soils . No difficulties were encountered

when this method was used routinely in the authors' laboratory

from 1968 until 1975, but several problems, including color

instability and decreased sensitivity, were observed during 1976.

Studies to account for these problems showed that they resulted

from impurities in some batches of phosphoric acid, but no clues

concerning the nature of these impurities could be obtained from

manufacturers or suppliers of phosphoric acid, and attempts to

find a commercial source of phosphoric acid that was consistently

free of these impurities proved unsuccessful. The purpose of this

communication is to describe a modified diacetyl monoxime method

 COLORIMETRIC DETERMINATION OF UREA 1165

of determining urea in soil extracts that permits use of phosphoric

acid containing the impurities that affect the method proposed by

Douglas and Bremner . The modified method is more exacting than

the original method, but it is more sensitive and precise.

COLORIMETRIC DETERMINATION OF UREA IN SOIL EXTRACTS

Reagents
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Potassium chloride-phenylmercuric acetate (2M KC1-PMA) solution.

Prepare as described by Douglas and Bremner .

Diacetyl monoxime (DAM) solution. Dissolve 2.5 g of diacetyl

monoxime (Fisher Scientific Co. , Chicago, IL) in 100 ml of dis-

tilled water.

Thiosemicarbazide (TSC) solution. Dissolve 0.25 g of thio-

semicarbazide (Eastman Organic Chemicals, Rochester, NY) in 100 ml

of distilled water.

Acid reagent. Add 40 ml of concentrated sulfuric acid (Reagent

A.C.S.) to 1,000 ml of 85% (w/w) phosphoric acid, dilute the mixture

to 2,000 ml with distilled water, and mix thoroughly.

Color reagent. Mix 50 ml of DAM solution with 30 ml of TSC

solution, and dilute the mixture to 1,000 ml with acid reagent.

Prepare this reagent immediately before use.

Standard urea N solution. Dissolve 0.4288 g of pure, dry

urea in 2M KC1-PMA solution in a 2,000 ml volumetric flask, dilute

the solution to volume with 2M KC1-PMA solution, and mix thoroughly.

This solution contains 100 pg of urea N per ml. Store it in a

refrigerator.
 1166 • MULVANEY AND BREMNER

Procedure

Pipette an aliquot (1-10 ml) of the soil extract containing

up to 100 yg of urea N into a 50-ml volumetric flask, make the

volume to 10 ml with 2M KC1-PMA solution, and add 30 ml of color

reagent. Swirl the flask for a few seconds to mix the contents,

place it in a water bath maintained at 85 + 0.5°C, and place a

lightproof cover on the bath. After 30 minutes, remove the flask

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from the water bath, cool it for 10 minutes in a bath of running

tap water (12-15°C), make the contents to 50 ml with distilled

water, and mix thoroughly. Then transfer about 10 ml of this

solution to a Klett-Summerson colorimeter tube (1.3-cm light path),

and measure its red color intensity with a Klett-Summerson

photoelectric colorimeter fitted with a green (no. 54) filter.

Calculate the urea N content of the extract by reference to a

calibration graph plotted from the results obtained with standards

containing 0, 10, 50, and 100 yg of urea N. To prepare this graph,

dilute 20 ml of the standard urea N solution to 200 ml with

2M KC1-PMA solution in a volumetric flask, and mix thoroughly.

Then pipette 0-, 1-, 5-, and 10-ml aliquots of this diluted standard

solution into 50-ml volumetric flasks, make the volume to 10 ml

by adding 2M KC1-PMA solution, and proceed as described for urea N

analysis of the soil extract.

All absorbance values reported were calculated from color

intensity measurements with the Klett-Summerson colorimeter (scale

reading x 0.002).
 COLORIMETRIC DETERMINATION OF UREA 1167

RESULTS AND DISCUSSION

The method described has been used extensively to determine

urea in extracts obtained by shaking soils with 2M KC1-PMA solution

as described by Douglas and Bremner , and no difficulties have

been experienced in its application to extracts of a wide variety

of soils. It gives no trace of red color when applied to extracts

of soils not previously treated with urea, and it gives quantitative

(99-100%) recovery of urea added to these extracts. Its precision

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is illustrated by Table 1, which shows the results of replicate

analyses of 0.5-10 ml aliquots of the diluted standard urea N

solution used to prepare the calibration curves.

TABLE 1

Precision of Method

Urea N added Urea N recovered (yg)*

(lig) Range Mean SD

5.0 4.9-5.0 5.0 0.05

10.0 9.9-10.0 10.0 0.06

25.0 24.9-25.1 25.0 0.06

50.0 49.9-50.1 50.0 0.08

75.0 74.9-75.1 75.0 0.10

100.0 99.8-100.0 99.9 0.12

Six analyses. SD, standard deviation.

 1168 MULVANEY AND BREMNER

The calibration curves obtained in the procedure described

show a linear relationship between urea N concentration and color

intensity, but they depend upon the source of the phosphoric acid,

and different samples of phosphoric acid from the same source give

different curves (Fig. 1 ) . It is essential, therefore, to include

urea N standards in each series of analyses.

The color developed in the procedure described is unstable,

but this is not a problem if color intensity measurements are per-

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formed shortly after color development because color fading is

negligible if the time between color development and color inten-

sity measurement does not exceed 60 minutes. This is illustrated

by Table 2, which also shows that the stability of the color obtained

.2 ° F (745160)
• F (753125) /
1.0 - ° F (774887)

f
• R /RflQRRfll /

0.8 - * M (KHJY) / 6

0.6 -

0.4 -

0.2
~ J/
r \ i I
0
) 10 50 100
UREA N. jjg

FIG. 1

Calibration curves obtained with different samples of phosphoric

acid. F, Fisher Scientific Co., Chicago, IL; B, J. T. Baker
Chemical Co., Phillipsburg, NJ; M, Mallinckrodt, Inc., Paris,
KY (lot numbers of products used are-shown in parentheses).
 COLORIMETRIC DETERMINATION OF UREA 1169

TABLE 2

Stability of Color*

Time after development Phosphoric acid

of color (minutes) F (774887) B (809868) M (KHJY)

0 0.990 0.960 1.020

30 0.990 0.960 1.020

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60 0.990 0.960 1.020

90 0.975 0.870 0.950

120 0.960 0.790 0.880

180 0.940 0.646 0.750

240 0.906 0.628 0.684

*Color obtained with 1 ml of solution containing 100 yg of urea N.

+
See Fig. 1 for identification of samples of phosphoric acid.

depends upon the sample of phosphoric acid used to prepare the

color reagent.

It is important that the flask used for color development be

cooled immediately after it is removed from the hot water bath be-

cause some loss of color occurs if the flask is not cooled rapidly

as specified.

Any colorimeter or spectrophotometer that permits color in-

tensity measurements at 500-550 nm can be used for the procedure

described. The maximum absorption of the color is at 527 nm. If

 1170 MULVANEY AND BREMNER

necessary, the sensitivity of the procedure can be increased by

use of Klett-Summerson colorimeter cells (4-cm light path) instead

of the colorimeter tubes specified.

The color reagent is unstable and should be prepared immed-

iately before use. The other reagents used are stable for several

weeks.

Nitrite interferes with the method described if the concentra-

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tion of nitrite N in the extract is more than five times the con-

centration of urea N, but interference by nitrite can be eliminated

very easily by the sulfamic acid technique described by Douglas and

Bremner .

ACKNOWLEDGMENT

This work was supported in part by the Tennessee Valley Authority.

REFERENCES

1. Journal Paper No. J-9449 of the Iowa Agr. and Home Econ. Exp.
Sta., Ames, Iowa. Project No. 2096.

2. E. A. Harre, W. H. Garman, and W. C. White. In R. A. Olson,

T. J. Army, J. J. Hanway, and V. J. Kilmer (eds.) Fertilizer
Technology and Use, 2nd edn., 27. Soil Science Society of
America, Madison, Wisconsin (1971).

3. O. P. Engelstad and R. D. Hauck. Crops & Soils 26, 11 (1974).

4. J. D. Beaton. Crops & Soils 30, 11 (1978).

5. L. A. Douglas and J. M. Bremner. Soil Sci. Soc. Amer. Proc.

34, 859 (1970).

6. J. M. Bremner and R. L. Mulvaney. In R. G. Burns (ed.) Soil

Enzymes, 149. Academic Press, London (1978).