Communications in Soil Science and Plant Analysis

ISSN: 0010-3624 (Print) 1532-2416 (Online) Journal homepage: http://www.tandfonline.com/loi/lcss20

A manual colorimetric procedure for measuring

ammonium nitrogen in soil and plant Kjeldahl
digests

W. E. Baethgen & M. M. Alley

To cite this article: W. E. Baethgen & M. M. Alley (1989) A manual colorimetric procedure for
measuring ammonium nitrogen in soil and plant Kjeldahl digests, Communications in Soil Science
and Plant Analysis, 20:9-10, 961-969, DOI: 10.1080/00103628909368129

To link to this article: https://doi.org/10.1080/00103628909368129

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 COMMUN. IN SOIL SCI. PLANT ANAL., 20(9410), 961-969 (1989)

A MANUAL COLORIMETRIC PROCEDURE FOR MEASURING

AMMONIUM NITROGEN IN SOIL AND PLANT
KJELDAHL DIGESTS

W. E. Baethgen
International Fertilizer Development Center
P.O. Box 2040, Muscle Shoals AL 35661

and

M. M. Alley
Agronomy Department
Virginia Polytechnic Institute and State University
Blacksburg, VA 24061

ABSTRACT

The measurement of NH4+-N in soil, and plant digests is one of the

greatest needs in laboratories conducting agricultural and
environmental research. Many laboratories do not have access to
automated equipment for colorimetric analysis of soil and plant
digests. The objective of this research was to modify an automated
colorimetric analysis procedure for determining NH4+-N in soil and
plant digests for manual use, and compare the proposed technique
with the standard distillation-titration technique. The modified

961

Copyright© 1989 by Marcel Dekker, Inc.

962 BAETHGEN AND ALLEY

procedure is based on the color reaction between NH4+- and a weakly

alkaline mixture of Na salicylate and a chlorine source in the
presence of Na nitroprusside. Wavelength scans indicated a very well
defined peak for determinations at 650 nm. Time scans showed that
color development in the manual procedure was rapid, 12 to 40
minutes depending on temperature, and that the color development
remained stable for at least 120 minutes. Regression analysis of the
results from 18 soil and 20 plant tissue sample determinations by
distillation-titration and the proposed method indicated NH 4 + -N
recoveries of 99% or higher. The results obtained using the
colorimetric procedure were very similar to the values obtained by
distil ling and titrating the digests for both soil and plant
samples as indicated by the large coefficients of determination (R2
= 0.99).

INTRODUCTION
Total N content of soil and plant tissue samples is generally
determined utilizing the Kjeldahl method (1). After digestion
NH4 + -N is frequently analyzed by steam distillation and titration
procedures (1). These procedures are simple to perform, require
relatively low cost equipment and produce reproducible results.
However, they demand considerable time and laboratory space. This
may be particularly inconvenient in laboratories where a large
number of soil and plant samples are analyzed and where automated
equipment or batteries of distillation units are not available.
Alternative procedures to reduce these problems are the use of
A MANUAL COLORIMETRIC PROCEDURE 963

ammonia electrodes (2, 3, 4) or by colorimetric determination of

NH4 + -N in the sample digests (5). However, the colorimetric
+
determination of NH4 -N proposed by Weatherburn (5) utilizes the
carcinogenic reagent phenol.
The objectives of this research were to modify an automated
colorimetric NHa + 'N analysis procedure for manual use, and to
compare the results obtained by the proposed technique with those
obtained by the standard distillation-titration technique.

MATERIALS
The soils utilized in this research were surface and subsurface
samples from the Coastal Plain, Piedmont, and Ridge and Valley
regions of Virginia with a wide range in total N content (6, 7).
Wheat tissue samples taken at different crop growth stages (7) from
N fertilization experiments were used as the plant samples. Soil and
plant samples were digested using the regular Kjeldahl method as
described by Bremner and Mulvaney (1), and replicate NH4 + -N
determinations were performed on all samples. A Beckman DU-7
spectrophotometer was used to determine the optimum wavelength for
the procedure and the minimum time needed to obtain full color
development in the samples.

METHOD
The proposed colorimetric procedure is a manual adaptation of an
automated procedure described by Wall et al. (8). The method is
based on the color reaction between ammonium and a weakly alkaline
964 BAETHGEN AND ALLEY

mixture of Na salicylate and a chlorine source, e.g., Na

hypochlorite (8) or dichloroisocyanurate (9). The rate and
intensity of the green color formed is greatly enhanced by the use
of Na nitroprusside. Sodium tartrate is added to eliminate the
precipitation of hydroxides of heavy metals which may be present in
the digests, especially if the catalyst mixture used in the
digestion contains heavy metals such as Cu, Se, or Hg.
Reagent Preparation
Working Buffer Solution (0.1 M Na2HPO/|. 5% Na-K tartrate. 5.4%
NaOH): Dissolve 26.8 g Na2HPC>4.7H2O in 600 mL of ammonium-free
water. Add 50 g Na-K tartrate, and 108 g 50% w/w solution of NaOH.
Dilute to 1 L with water.
Na salicylate-Na Nitroprusside Solution C15% - 0.03%V. Dissolve
150 g Na salicylate and 0.30 g Na nitroprusside in 1 L of water.
Store in a light-resistant bottle.
Na Hypochlorite Solution: Dilute 6 mL 5.25% Na hypochlorite
solution to 100 mL with water. This solution must be prepared fresh
daily.
Diluent: Dissolve 22 g catalyst mixture used for the Kjeldahl
digestion (100:10:1 mixture of ^SO^CuSO^Se) as described by
Bremner and Mulvaney (1) in 1 L of 1.1 M H2SO4.
Nitrogen standard solutions: Dissolve 4.715 g dry (NH4)2SO4 in
1 L of diluent to prepare a stock solution containing 1000 mg N/L.
Using diluent prepare standard solutions containing 5.0, 10.0, 15.0,
30.0, 40.0, and 50.0 mg N/L.
A MANUAL COLORIMETRIC PROCEDURE 965

Description of the Procedure:

Digestion:
Digest 500 mg plant tissue, or 1000 mg soil, in 3 mL
concentrated H2SO4 (18 M) adding 1.1 g catalyst mixture (100:10:1 of
K2SO4:CuSO4:Se) (1). After the digestion is completed (1), dilute
digest with water to a final volume of 50 mL.

Colorimetric Determination of Ammonium-N:

Dilute the Kjeldahl digests with water to obtain solutions
containing 1 to 50 mg N/L. The plant digests in this research were
diluted 50 times and the soil digests were diluted 5 times using an
automatic dispenser-diluter. Add 5.5 mL working buffer solution to
a 1 mL aliquot of the diluted digest in a test tube and stir with a
vortex mixer. Add 4 mL Na salicylate-Na nitroprusside solution and
mix again. Add 2 mL Na hypochlorite solution and mix thoroughly.
The reagents must be added in the stated order to avoid the
formation of solid residues. Let stand for 45 minutes at 25°C (or 15
minutes at 37°C) to ensure complete color development. Read
absorbance in a spectrophotometer using a wavelength of 650 nm. Each
sample should be stirred with the vortex mixer immediately before
the colorimetric determination.

RESULTS AND DISCUSSION

Wavelength scans were performed to determine the optimum

wavelength for the procedure and a very well defined peak was found
966 BAETHGEN AND ALLEY

at 650 nm (Fig. 1). Time scans were then run to determine the
minimum period of time (t) needed to attain full color development.
The time scans were performed at 25°C and 37°C. The t values for
these temperatures were 40 and 12 minutes, respectively (Fig. 2),
and the developed color was stable for at least 120 minutes (data
not shown).
Eighteen soil and 20 plant tissue digests were assayed
comparing determined NH4+-N results using the proposed colorimetric
procedure with those obtained by means of the steam distillation -
titration procedure.
A simple linear regression with intercept equal to zero was
adjusted using the results of the distillation-titration procedure
as the dependent variable and the results of the colorimetric method
as • the independent variable. The estimated regression coefficients
indicated that the NH4 + +-N recovery by the colorimetric procedure
was 99% or higher as compared to the distillation-titration
procedure. The results obtained using the colorimetric procedure
were very similar to the ones obtained by distilling and titrating
the digests for both soil and plant samples as indicated by the
large coefficients of determination (R.2 = 0.99, Fig. 3a and 3b).
The catalyst mixture used in the Kjeldahl digestion (100:10:1
mixture of ^SO^CuSO^Se) (1) often produces some degree of
turbidity in the sample digests. Therefore, the digests should be
filtered or let stand overnight and the aliquots for colorimetric
determination taken carefully. Turbidity problems can be eliminated if
the digestion is performed using hydrogen peroxide instead of the
A MANUAL COLORIMETRIC PROCEDURE 967

0.48-
\ V

0.36-
/ \

j / f
\

S 0.24- / \

/ \

0.12- [/ \

0.00-
550 S90 &M era 710 750
Wavelength (nm)

Figure 1. Wavelength scan used to determine optimum wavelength for

colorimetric ammonium determination.

.500 n

— 25-c
— 37-c
30 60 90
Time (minutes)
Figure 2. Time scans performed at two temperatures (25°C and 37°C)
used to determine the minimum period of time needed to
attain full color development.
 968 BAETHGEN AND ALLEY

a) Soil digests b) Plant digests

50-

lu 40
eo l / c«
3000- /
. - OO
~ - 30H
/ <0 00

Ik 1500-
20H
b oo / y = 1.015 x y = 1.002 x
rff R 2 - 0.999 ^ ioH R2 . 0.999
0- r 0
1500 3000 4500 10 20 30 40 50
Colorimetric Colorimetric
(mg N / Kg soil) (g N / Kg tissue)
Figure 3. Relationship between the distillation - titration procedure
and the proposed colorimetric procedure for soil (a) and
plant (b) sample digests.

catalyst mixture (8). This digestion procedure is faster and the

resulting digests do not contain any precipitates. The diluent
described in the methodology section should be modified in order to
ensure that the N standard solutions contain the same components and
concentrations as the sample digests to be measured.

CONCLUSION
The proposed colorimetric procedure is precise, fast, simple,
and does not require a large laboratory space or special laboratory
equipment. The technique is particularly suited to research
laboratories in developing countries where automated equipment is
generally not available. The results indicated a mean NH4 + +-N
recovery of at least 99% compared to the distillation - titration
technique, and a close agreement between the two methods.
A MANUAL COLORIMETRIC PROCEDURE 969

Furthermore, the proposed colorimetric procedure does not include

the carcinogenic phenol reagent of other commonly used colorimetric
techniques.

REFERENCES

1. Bremner, J.M., and C.S. Mulvaney. 1982. Nitrogen-total, pp

595-624. IN: A.L. Page et al. (ed) Methods of soil analysis,
Part 2, 2nd ed. Am. Soc. Agron., Madison, WI.
2. Bremner, J.M., and M.A. Tabatabai. 1972. Use of an ammonia
electrode for determination of ammonium in Kjeldahl analysis of
soils. Comm. Soil Sci. Plant Anal. 3:159-165.
3. Eastin, E.F. 1976. Use of ammonia electrode for total nitrogen
determination in plants. Comm. Soil Sci. Plant Anal. 7:477-481.
4. Powers, R.F., D.L. Van Gent, and R.F. Townsend. 1981. Ammonia
electrode analysis of nitrogen in microKjeldahl digests of
forest vegetation. Comm. Soil Sci. Plant Anal. 12:19-30.
5. Weatherburn, M.W. 1967. Phenol-hypochlorite reaction for
determination of ammonia. Anal. Chem. 39:971-974.
6. Baethgen, W.E., and M.M. Alley. 1987. Nonexchangeable ammonium
contribution to plant available N in Virginia soils. Soil Sci.
Soc. Amer. J. 51:110-115.
7. Baethgen, W.E., and M.M. Alley. 1988. Optimizing soil and
fertilizer nitrogen use for intensively managed winter wheat.
II. Critical levels and optimum N fertilizer rates. Soil Sci.
Soc. Amer. J. (in press)
8. Wall, L.L., C.W. Gehrke, T.E. Neuner, R.D. Cathey, and P.R.
Rexroad. 1975. Total protein nitrogen: evaluation and
comparison of four different methods. J. Assoc. Off. Anal.
Chem. 58:811-817.
9. Crooke, W.M., and W. E. Simpson. 1971. Determination of
ammonium in Kjeldahl digests of crops by an automated procedure.
J. Sci. Food Agric. 22:9-10.