Improving the Berthelot Reaction for Determining Ammonium
in Soil Extracts and Water
E. D. Rhine, G. K. Sims,* R. L. Mulvaney, and E. J. Pratt
ABSTRACT
Colorimetric methods based on the Berthelot reaction are used
widely for quantitative determination of NH4-N in biological and
environmental samples. Studies to evaluate phenol and salicylate,
the most commonly used chromogenic substrates, revealed minor
interferences by metallic cations, whereas up to a threefold shift in
absorbance was observed with 38 diverse N-containing organic com-
pounds. Interferences differed markedly between phenol and salicy-
late. The possibility of a simple correction was precluded by the fact
that interferences were both positive and negative, and depended on
the temperature during color development and the concentration of
NHj-N. Fourteen compounds were evaluated as alternatives to phenol
and salicylate, of which the Na salt of 2-phenylphenol (PPS) proved
to be the most promising. Using PPS, macro- and microscale batch
methods and an automated flow-injection method were developed.
These methods are simple, convenient, and sensitive. Using the PPS
microscale method, for which the limit of detection is 0.17 mg NH4-N
L"1, recovery of NHf-N added to soil extracts ranged from 98 to
104%, with a coefficient of variation of 1.4 to 2.7%. As with phenol
and salicylate, precipitation of metal hydroxides was observed. Precip-
itation was controlled by chelation with citrate rather than ethylenedi-
aminetetraacetic acid (EDTA), which suppressed color development
by preventing monochloramine formation. Compared with Berthelot
methods that use phenol or salicylate, interference by amino acids
was decreased by up to 10-fold. Interference by other organic N
compounds was virtually eliminated.
C OLORIMETRIC METHODS are commonly used to deter-
mine inorganic N in soil extracts and water sam-
ples. These methods are simple and sensitive (Keeney
and Nelson, 1982), and have been widely employed in
the design of automated analyzer systems (Mulvaney,
1996). Colorimetric analyses of NH4 are usually based
on the Berthelot method, which involves reaction of
NH4 as monochloramine with hypochlorite and phenol,
or a phenolic compound having an unsubstituted para-
position, under alkaline conditions (Searle, 1984). Phe-
E.D. Rhine, R.L. Mulvaney, and E.J. Pratt, Dep. of Natural Resources
and Environmental Sciences, Univ. of Illinois; and G.K. Sims, USDA-
ARS, 1102 S. Goodwin Ave., Urbana, IL 61801. Received 14 April
1997. *Corresponding author (gk-sims@uiuc.edu).
Published in Soil Sci. Soc. Am. J. 62:473-480 (1998).
nol has been used more extensively than other phenolic
compounds, but sodium salicylate also has been used
widely because it is less toxic than phenol and is a more
convenient reagent. The reaction product has a blue or
blue-green color, the intensity of which is presumably
related to the concentration of NH4 within the sample.
Gentle heating (37^tO°C) is often employed to reduce
the period for color development and increase sensitiv-
ity (Keeney and Nelson, 1982; Mulvaney, 1996).
Color formation by the Berthelot reaction is strongly
influenced by pH. The optimum pH ranges from 10.5
to 13, depending on the chromogenic substrate used
(Searle, 1984). These strongly alkaline conditions pro-
mote precipitation of metal hydroxides and thereby
complicate the application of the Berthelot reaction for
NH4-N analysis of environmental samples. Chelating
agents, such as EDTA or citrate, have commonly been
employed to reduce interference by Ca and Mg in sea-
water (Roskam and de Langen, 1964) and soil extracts
(Keeney and Nelson, 1982). Besides causing precipita-
tion, some metals, including Cu and Hg, interfere with
color formation when present in high concentration, as
can occur in analysis of Kjeldahl digests.
The Berthelot reaction is not entirely specific for NH4.
In addition to NH4, color is obtained with a variety
of organic compounds that contain N, including amino
acids, amines, and amides (Searle, 1984). This color is
a potential source of interference in analysis of soil
extracts, which generally contain organic N, and serious
interference has been reported with soils high in organic
C (White and Gosz, 1981) and with those that have
been fumigated or dried (Burton et al, 1989). Organic
interference has been attributed to the release of NH3
following hydrolysis under the strongly alkaline condi-
tions inherent in the Berthelot reaction. Attempts to
control organic N interference by reducing reaction tem-
perature or pH have produced mixed results (White
and Gosz, 1981; Rowland, 1983). Steam distillation has
Abbreviations: CV, coefficient of variation; EDTA, ethylenediamine-
tetraacetic acid; LSD, least significant difference; PPS, 2-phenylphe-
nol, Na salt tetrahydrate; SD, standard deviation.
474 SOIL SCI. SOC. AM. J., VOL. 62, MARCH-APRIL 1998

Table 1. Analyses of soils.t

Soil
Series Subgroup Total N Organic C Sand Silt Clay
-i
Plainfield mixed, mesic Typic Udipsamment 7.6 0.23 3.0 946 41 13
Cisne fine, montmorillonitic, mesic Mollic Albaqualf 6.3 1.08 11.0 186 661 153
Harpster fine-silty, mesic Typic Calciaquoll 7.2 2.87 28.3 49 565 386
Drummer fine-silty, mixed, superactive, mesic 6.2 2.77 33.2 52 606 342
Typic Endoaquoll
Houghton euic, mesic Typic Medisaprist 7.6 7.93 126.3 - - -
t Analyses for pH, total N, organic C, and texture were performed as described by Mnlvaney and Kurtz (1982).
been proposed as a means of avoiding organic interfer- Mechanical Pipettors. A multichannel pipettor, adjustable
ences in analysis of soil extracts (Burton et al., 1989); to 125 U.L with coefficient of variation (CV) <2%, was used
however, NH4 in the distillate is more readily deter- for reagent dispensing. An adjustable pipettor (10-100 pX)
mined by acidimetric titration than by colorimetry. with CV <0.4% was used for dispensing samples.
Microplate Reader. A Ceres Model 900 Microplate Work-
This project originated from the lack of an effective
station (BioTek Instruments, Winooski, VT) was used to mea-
means of controlling organic interferences in analysis sure absorbance at 660 nm.
of soil extracts by existing Berthelot methods. Based Vortex Mixer. A Vortex-Genie 2 shaker fitted with a mi-
on the assumption that these interferences originate croplate adapter (Scientific Industries, Bohemia, NY) was
through hydrolysis of organic N, studies were conducted used.
to identify an alternative to phenol and salicylate that
would allow color development at a lower pH. Of the Reagents
compounds tested, PPS was selected as the most promis-
ing; however, further studies indicated a low reaction pH 2-Phenylphenol-Nitroprusside Reagent. 2-Phenylphenol
to be impractical. Eventually, PPS was used to develop (3.22 g) and 0.015 g of sodium nitroprusside [Na2Fe(CN)5-
NO5H2O] were dissolved in 100 mL of deionized water in a
microplate, macroscale, and automated flow-injection volumetric flask.
methods for NH4 analysis of soil extracts. The primary Citrate Reagent. Five grams of trisodium citrate (Na3-
objectives of our study were to develop and evaluate C6H5O7-2H2O) were dissolved in =80 mL of deionized water
these methods, and to compare PPS with phenol and in a 100-mL volumetric flask. The pH was adjusted to 7.0 by
salicylate in regard to interference by a wide variety of the addition of 0.1 M HC1 and the solution was diluted to
organic N compounds. 100 mL.
Buffered Hypochlorite Reagent. One gram of Na3PO4 was
MATERIALS AND METHODS dissolved in approximately 80 mL of deionized water in a 100-
mL volumetric flask. Then 10 mL of sodium hypochlorite
Soil Extracts solution (0.7 M NaOCl; household bleach) was added, the pH
The soils used (Table 1) were surface (0-15 cm) samples was adjusted to 13.0 with 2 M NaOH, and the solution was
of five soils from Illinois selected to obtain a wide range in diluted to 100 mL.
physicochemical characteristics. The Plainfield (mixed, mesic Standard Ammonium-Nitrogen Solutions. Exactly 0.9346 g
Typic Udipsamment), Cisne (fine montmorillonitic, mesic of (NH4)2SO4 was dissolved in 1 L of deionized water in a
Mollic Albaqualf), Harpster (fine-silty, mesic Typic Calci- volumetric flask to produce a concentrated standard con-
aquoll), and Drummer (fine-silty, mixed, superactive, mesic taining 200 mg of NH4-N L"1. This solution was stored in a
Typic Endoaquoll) soils were collected from cultivated fields refrigerator. Working standards were prepared daily by dilut-
under corn (Zea mays L.) and soybean [Glycine max (L.) ing the concentrated standard with deionized water to obtain
Merr.] production; Houghton (euic, mesic Typic Medisaprist) concentrations of 0, 0.5, 1.0, 2.0, 5.0, 10.0, and 20.0 mg
is a waterlogged organic soil. Before use, each sample was air NH4-N L-1.
dried and crushed to pass a 2-mm screen. Soil extracts were
obtained by shaking 10 g of soil with 100 mL of 2 M KC1 for Sources of Reagents
1 h in a 125-mL polyethylene bottle. The resulting suspensions
were filtered through Whatman no. 42 filter paper under vac- The NaOCl specified was manufactured by Clorox Co.,
uum (Mulvaney, 1996). Extracts were stored at 5°C for no Oakland, CA. The other reagents specified were obtained
longer than 6 mo. from Aldrich Chemical Co., Milwaukee, WI.

Microplate Method Procedure

Materials
Ammonium Assay Plates. Flat-bottom polystyrene assay
plates (96-well) with lids (cat. no. 25880-96, Corning Glass
Works, Corning, NY) were used.1 Each well has a capacity of
300 (jiL.
1
Names are necessary to report factually on available data; how-
ever, the USDA neither guarantees nor warrants the standard of the
product, and the use of the name by USDA implies no approval of the
product to the exclusion of other products that may also be suitable.
A 50-p-L aliquot of soil extract or standard NH4-N solution
was pipetted into individual wells of an assay plate. Fifty micro-
liters of citrate reagent were then added and allowed to react
for at least 1 min. Fifty microliters of the PPS-nitroprusside
reagent were added to the wells, followed by 25 jjiL of the
buffered hypochlorite reagent, and then 100 \L~L of deionized
water. After attaching a lid, the plate was gently agitated for
30 s on a vortex mixer, and was then allowed to stand for
45 min to ensure complete color development. The lid was
removed from the plate, and absorbance was measured with
 RHINE ET AL.: IMPROVING THE BERTHELOT REACTION FOR DETERMINING AMMONIUM
a microplate reader at 660 nm. Absorbance measurements
were calibrated by regression analysis of data obtained with
the standards. The regression equation was used to convert
absorbance values for soil extracts to NH4-N concentrations.
Macroscale Method
Materials
Absorbance measurements at 660 nm were performed using
a spectrophotometer equipped to provide a 1-cm light path.
Reagents
2-PhenyIphenoI-Mtroprusside Reagent. In a 250-mL volu-
metric flask, 7.32 g of PPS and 0.034 g of Na nitroprusside
were dissolved in deionized water and brought to volume.
Citrate Reagent. Trisodium citrate (11.36 g) was dissolved
in approximately 180 mL of deionized water in a 250-mL
volumetric flask. The pH was adjusted to 7.0 by the addition
of 0.1 M HC1 and the solution was made to volume with
deionized water.
Buffered Hypochlorite Reagent. Trisodium phosphate
(2.27 g) was dissolved in approximately 180 mL of deionized
water. Then 22.5 mL of household bleach was added, the pH
was adjusted to 13.0 with 2 M NaOH, and the solution was
diluted to 250 mL.
Standard Ammonium-Nitrogen Solution. A working stan-
dard NH4-N solution (2 mg L"1) was prepared by diluting
2 mL of the above specified concentrated standard solution
to 200 mL in a volumetric flask with 2 M KC1.
Procedure
A 5-mL aliquot of soil extract was added to a 50-mL volu-
metric flask. Five milliliters of the citrate reagent were then
dispensed into the flask, and the flask was swirled briefly.
After 1 min, 5 mL of the PPS-nitroprusside reagent were
added, and the volume was brought to =40 mL with deionized
water. Color formation was initiated by the addition of 3 mL
of the buffered hypochlorite reagent, after which the volume
was immediately brought to 50 mL with deionized water, and
the contents of the flask were mixed thoroughly. The flask was
allowed to stand for 45 min at room temperature. Absorbance
measurements were made at 660 nm against a reagent blank
solution. To calibrate these measurements, analyses were per-
formed on standards containing 0, 2.0, 4.0, 6.0,10.0, and 20.0
jig of NH4-N. The standards were prepared by pipetting into
six 50-mL volumetric flasks the same volume of extracting
solution (or water, depending on the sample matrix) as the
aliquot of soil extract taken for analysis, and adding 0, 1.0,
2.0, 3.0, 5.0, or 10.0 mL of the working standard (NH4)2SO4
solution (2 mg of NH4-N L~').
Automated Analyzer Method
Materials
A QuickChem Automatic Flow Injection Ion Analyzer (La-
chat Instruments, Milwaukee, WI) equipped with the appro-
priate manifold and interference filter for NFL-N analysis by
QuickChem Method no. 12-107-06-2-A was used to develop
an automated method based on PPS.
Reagents
Potassium Chloride Carrier and Standards Diluent. A 2 M
KC1 solution was prepared by dissolving 150 g of the reagent-
grade material in 1 L of deionized water.
475
2-Phenylphenol-Nitroprusside Reagent. In 1L of deionized
water in a volumetric flask, 32.23 g of PPS and 0.15 g of Na
nitroprusside were dissolved.
Citrate Reagent. Fifty grams of trisodium citrate were dis-
solved in approximately 800 mL of deionized water in a 1-L
volumetric flask. The pH of this solution was adjusted to 7.0
by the addition of 1 M HC1 and the volume was brought to
1 L with deionized water.
Buffered Hypochlorite Reagent. In a 1-L volumetric flask,
10 g of Na3PO4 were dissolved in approximately 800 mL of
deionized water. After adding 100 mL of household bleach,
the pH was adjusted to 13.0 with 2 M NaOH, and the solution
was diluted to 1 L with deionized water.
Standard Ammonium-Nitrogen Solutions. The concen-
trated standard solution (specified above) was diluted with
2 M KC1 to obtain NH4-N concentrations of 0.1, 0.25,0.5,1.0,
5.0, 10.0, and 20.0 mg L~'.
Degassing. All reagents except the buffered hypochlorite
and NH4-N standards were degassed with He for 1 min.
Operating Parameters
Analyses followed a 64-s cycle, after which the system was
purged with carrier fluid for 30 s. The response of the detector
at 660 nm was integrated for 38 s, beginning 26 s after injection.
The peristaltic pump was operated to obtain a flow rate of 35
mL min"1. A 70-cm sample loop was used, which provided a
364-(iL injection volume.
RESULTS AND DISCUSSION
Evaluation of Phenol and Salicylate
As noted above, the Berthelot reaction is not entirely
specific for NH4. This is illustrated by Fig. 1 and 2, which
summarize effects of the 38 organic and five inorganic
compounds listed in Table 2 on NH4-N measurements
using either phenol or salicylate as the chromogenic
reagent. With each reagent, interference tests were car-
ried out by heating at 37°C (Fig. 1) or at 25°C without
heating (Fig. 2), and at each temperature these tests
were done in triplicate with or without the addition of
NH4-N. To accommodate the large number of measure-
30
.T— 25
;B
'-J 20
? ' 5
x* 10
Z 5
I:
I- -10
jfl -15
S 10 15 20 25 30 35 40 5 10 15 20 25
REFERENCE NUMBER FOR INTERFERING COMPOUND
Fig. 1. Effects of the organic and inorganic compounds listed in Table
2 on color development by a microscale batch technique using (A,
B) phenol or (C, D) salicylate at 37°C; NH4-N concentration in
water = (A, C) 0 or (B, D) 10 mg L"1. Least significant difference =
0.194 mg NRr-N L~'.
476 SOIL SCI. SOC. AM. J., VOL. 62, MARCH-APRIL 1998

or natural water, and studies showed very little, if any,

111
EC
1C
111
oc
10 15 20 25 30 35 40 10 15 20 25
REFERENCE NUMBER FOR INTERFERING COMPOUND
Fig. 2. Effects of the organic and inorganic compounds listed in Table
2 on color development by a microscale batch technique using (A,
B) phenol or (C, D) salicylate at 25°C; NH4-N concentration in
water = (A, C) 0 or (B, D) 10 mg L~*. Least significant difference =
0.194 mg NHj-N L '.
ments involved (1080), color development was per-
formed by a microscale batch technique, based on the
salicylate method of Sims et al. (1995) or a modified
version of this method in which phenol, at the same
molar concentration, was substituted for salicylate. The
data are expressed in terms of an absolute deviation
from the actual NH4-N concentration (0 or 10 mg L"1).
Interferences were both positive and negative, and
depended on the temperature during color development
and the concentration of NH4-N. Heating, commonly
employed to promote color development, exacerbated
interference by most of the substances tested. Organic
interferences tended to be more serious than inorganic
interferences. Similar results were obtained when se-
lected interference tests were repeated using macroscale
format. Lower interferences were observed using an
automated flow-injection system when salicylate was
used (Fig. 3a and 3b). The latter finding can be attrib-
uted to incomplete color development, because a sub-
stantial increase in interference, and also higher sensitiv-
ity, was observed when the reaction time was extended
by adding 92 cm of heated tubing (Fig. 3c and 3d).
Despite the use of citrate as a chelating agent, sub-
stantial precipitation occurred with high concentrations
of Ca (2 g L"1) or Mg (1 g L*1) and usually led to
a small positive interference. Such concentrations are
unlikely to be encountered in the analysis of soil extracts
iB
evidence of precipitation when NH4-N analyses were
performed with up to 500 mg L"1 of Ca, or with up to
250 mg L"1 of Mg (data not reported). Iron concentra-
tions up to 20 mg LT1 had a negligible effect on NH4-N
analyses, whereas serious interference resulted when a
yellow color was formed with 50 to 500 mg Fe L"1,
which greatly exceeds the concentration of Fe in soil
extracts or natural water. Minimal interference was ob-
served with NO3 or NO2 (100 mg N L"1). This finding
differs from previous work by Hampson (1977) and
Willis et al. (1993), which indicated substantial interfer-
ence by NO2 when colorimetric analyses are performed
at low concentrations of NH4-N.
Of the organic compounds tested, interferences were
30 35 40
observed with purines and amides, particularly in the
presence of NH4, but the most serious interferences
occurred with amino acids. This finding was to be ex-
pected, since amino acids were used as reactants in early
investigations into the mechanism of the Berthelot reac-
tion (Tarugi and Lenci, 1912). Interference by amino
acids has commonly been attributed to hydrolysis, yield-
ing NH3, under strongly alkaline conditions (Searle,
1984). In our study, no release of NH4-N was detected
when amino acids were heated at 45 to 50°C for 3.5 h
under conditions similar to those employed in the Ber-
thelot reaction (pH 13), using a modified version of the
diffusion method described by Khan et al. (1997). The
latter finding suggests that the positive interferences
observed with amino acids originated because of a reac-
tion between the amino group and the reagents em-
ployed for color formation.
Examination of Fig. 1 and 2 reveals a marked differ-
ence between phenol and salicylate in regard to the
nature and extent of interference by many of the organic
compounds tested. Of particular interest is the prepon-
derance of negative interferences observed with phenol
compared with salicylate. This finding is consistent with
previous reports of negative interference by amino com-
pounds in phenol methods based on the Berthelot reac-
tion (e.g., Forgan-Smith et al., 1976; Ngo et al., 1982).
According to Ngo et al. (1982), amino groups react
readily with quinonechloramine intermediates in this
reaction, forming 2,5-bis(alkylamino)quinonechlora-
mines that do not react with monochloramine and
thereby inhibit color formation. This mechanism cannot
account for the results obtained with salicylate.
Figures 1 and 2 preclude the possibility that a simple
correction can be made to account for organic interfer-

Table 2. Numerical key to organic and inorganic compounds evaluated for interference in NH4-N analysis.
Organicf
Amino acids alanine (1), arginine (2), aspargine (3), aspartic acid (4), cystine (5), glutamic acid (6), glycine (7), histidine (8), isoleucine
(9), leucine (10), lysine (11), methionine (12), phenylalanine (13), proline (14), serine (15), threonine (16), tryptophan
(17), valine (18)
Purines adenine (19), gnanine (20), xanthine (21)
Pyrimidines cytosine (22), thymine (23), uracil (24)
Amides biuret (25), glutamine (26), nicotinamide (27), urea (28)
Hydroxypyridines 2-hydroxypyridine (29), 3-hydroxypyridine (30), 4-hydroxypyridine (31)
Miscellaneous casein (32), chitin (33), galactose (34), gelatin (35), glucosamine (36), nicotinic acid (37), pyridine (38)
Inorganic* CaCI2-2H2O (39), FeCI3 (40), MgCl2 (41), NaNO2 (42), KNO3 (43)
t Compound concentration = 10 mg of N L"1.
t Compound concentrations = 2 g of Ca L ' as CaCI2-2H2O,10 mg of Fe L ' as FeCI3,1 g of Mg L~ as MgO2,100 mg of N L ' as NaNO2 or KNO3.
 RHINE ET AL.: IMPROVING THE BERTHELOT REACTION FOR DETERMINING AMMONIUM 477

30
25 B
20
15
10
5
D)
0
-5
I-
-10
UJ -15
IS -20
LLI
cc 30
ID 25
CO
< 20
LU 15
10
5
DC 0
O -5
OC
EC -10
LU -15
-20
6 9 12 19 24 2S 30 39 42 1 6 9 12 19 24 26 30 39 42
REFERENCE NUMBER FOR INTERFERING COMPOUND
Fig. 3. Effects of selected organic and inorganic compounds listed in Table 2 on color development by an automated flow-injection system using
salicylate, (A, B; least significant difference = 0.154 mg NHr-N L ') before and (C, D; least significant difference = 0.194 mg NILr-N L"1)
after modification to increase reaction time; NH,|-N concentration = (A, C) 0 or (B, D) 10 mg L"1.

ences in Berthelot methods using either phenol or salicy- The monochloramine then reacts with PPS, presumably
late. Besides varying widely in magnitude and direction, forming a quinonechloramine (II). An indophenol dye
these interferences also depended on the presence or (III) was identified by mass spectroscopic analysis of
absence of NH4. The dramatic differences between sal- the neutral form (IV, mlz 353), and is the expected
icylate and phenol suggest that the most practical ap- product when II couples with excess PPS under strongly
proach to reducing organic interferences in NH4-N anal- alkaline conditions.
yses by the Berthelot reaction involves the choice of a A further advantage of PPS over phenol or salicylate
phenolic compound as the chromogenic substrate. More is that color formation occurs across a wider pH range.
than 65 substituted phenols have been used to form A study to compare these reagents showed that color
color by this reaction (Soloway and Santoro, 1955). is obtained with PPS down to a pH of 5, whereas no
color was detected below pH 8.5 with phenol and below
Development of Proposed Berthelot Methods 11.5 with salicylate. Subsequent work showed a low pH
to be impractical for color development with PPS, owing
Besides phenol and salicylate, the 14 compounds to the appearance of an immiscible red liquid and pre-
listed in Table 3 were evaluated for reactivity in forming cipitation of PPS. The red chromophore was shown to
color by the Berthelot reaction. Of the compounds be the neutral form of the indophenol dye. Protonation
tested, nine were found to give good sensitivity in previ- of the dye leads to a drastic decrease in solubility, and
ous evaluations in the chemical literature. The re- thereby shifts the equilibrium to favor the insoluble
maining five were tested as potential reactants that species. An increase in pH promoted dissociation to the
would be nontoxic and nonvolatile. In each case, color soluble (blue) form and thereby slowed accumulation
development was carried out at pH 8 to 13 so as to
optimize reactivity. Table 3. Limiting properties of chromogenic substrates evaluated
Most of the compounds studied proved to have very for use in the Berthelot reaction at pH 8 to 13.
limited potential for use in the Berthelot reaction, owing
Compound Limitation Reference
to low solubility in water, minimal reactivity in forming
color, or the presence of an offensive odor. The most 2-Chlorophenol volatility, hazard Soloway and Santoro, 1955
3-Chlorophenol volatility, hazard Soloway and Santoro, 1955
promising chromogenic substrate was PPS. Unlike the 2-Cresol volatility, hazard Soloway and Santoro, 1955
acid form of this compound that was used by Yamaguchi 3-Cresol volatility Soloway and Santoro, 1955
and Machida (1968), PPS is readily soluble in water. 2,6-Dimethoxyphenol reactivity
3,5-Dimethylphenol reactivity
Like salicylate, PPS is neither toxic nor caustic, and the Guaiacol solubility, hazard Yamaguchi and Machida,
reagent is supplied in the form of flaky crystals that are 1968
8-Hydroxyquinaldine solubility Morita and Kogure, 1970
easily weighed and transferred. The reaction of NH4 1-Naphthol reactivity Morita and Kogure, 1963
with PPS proceeds more rapidly than with either phenol 2-Naphthol reactivity
or salicylate, forming an intense blue color. PPSt none
2-Phenylphenol solubility Yamaguchi and Machida,
The reactions involved in color formation probably 1968
proceed according to the sequence indicated by Steps 3-Phenylphenol solubility, reactivity
A to C in Fig. 4. The first step, yielding monochloramine Thymol volatility, reactivity Soloway and Santoro, 1955
(I), is well established in the literature (Searle, 1984). t PPS = 2-phenylphenol, Na salt tetrahydrate.
478 SOIL SCI. SOC. AM. J., VOL. 62, MARCH-APRIL 1998

(A) NH3 + ocr NH2CI Table 4. Effects of solution pH on color development by the PPS
microplate method.t
(I)
NHr-N added (mg
pH during
color development 10 20
(B) — Cl
—— Absorbance ——
11.0 0.079 0.698 1.476 2.978
11.5 0.082 0.747 1.481 2.986
12.0 0.086 0.794 1.653 2.997
(II) 12.5
13.0
0.090
0.087
0.838
0.852
1.748
1.762
3.095
3.270
t PPS = 2-phenylphenol, Na salt tetrahydrate.
t Data reported are the mean of three replications using aqueous solutions.
Least significant difference = 0.112.

prusside were found to reduce the intensity of the blue

-Cl
Fig. 4. Reactions involved in color development by 2-phenylphenol,
Na salt tetrahydrate (PPS) methods: (A) monochloramine (I) for-
mation; (B) quinonechloramine (II) formation; (C) coupling reac-
tion to form dissociated (III) and neutral (IV) indophenol dye.
of the insoluble (red) product. A higher pH also reduced
precipitation of PPS, and precipitation was eliminated
completely when analyses were performed at pH 11 to
13. Table 4 shows that, within this range, a progressive
increase occurs in absorbance with increasing pH, and
in the methods described, a pH of 13 was adopted so
as to maximize sensitivity. A higher pH is not recom-
mended because excessive color development reduces
the dynamic range.
To enhance sensitivity, the Berthelot reaction is usu-
ally carried out in the presence of a catalyst. Other
reagents have been employed for this purpose, but so-
dium nitroprusside is preferred because it is a more
effective catalyst and has been widely employed in Ber-
thelot methods to reduce the period for color develop-
ment and increase color stability (Searle, 1984). The
same catalyst is used in the methods described, at a
concentration of 0.6 mM. Lower concentrations of nitro-
color formed, whereas higher concentrations produced
a green color and increased the absorbance of the blank.
Studies to compare different concentrations of PPS
in the reaction showed the concentration specified for
the PPS-nitroprusside reagent (0.12 M) to be optimal.
Sensitivity was sacrificed with a lower concentration.
Higher concentrations promoted precipitation of PPS
with no gain in molar absorptivity.
The blue color obtained in the methods described
exhibits maximal absorbance at 665 nm, and the highest
sensitivity is achieved when measurements are made at
this wavelength. Absorbance measurements may not be
possible at 665 nm in cases where wavelength selection
is accomplished through the use of a colored filter rather
than a monochromator, as with most microplate readers
and automated analyzers. In such cases, a filter should
be used that provides the closest available wavelength,
within the range of 650 to 680 nm.
Absorbance was nearly maximized within 15 min for
samples containing up to 5 mg of NH4-N L-1, but a 45-
min period is specified in the macro- and microscale
methods described to ensure complete color develop-
ment with up to 20 mg of NH4-N L'1 at 25°C (Table
5). The color is stable for about 3 h when the microplate
is left uncovered, but thereafter a decrease occurs in
absorbance because of evaporation, which reduces the
optical path length and promotes formation of a red
film from the presence of the neutral indophenol dye.
The absorbance is stable for at least 12 h when the
microplate is covered with a lid as recommended.
A chelating agent is commonly employed to reduce
interference by metallic cations in Berthelot methods
for NH4-N analysis of soil extracts. Three such agents
(tartrate, EDTA, and citrate) were evaluated for use

Table 5. Rate of color development with the PPS microplate method.t

Period of development (min)f
NHr-N 15 30 45 60 75 90 105 120
it_ ,_
0 0.097 0.099 0.099 0.100 0.099 0.100 0.100 0.100
0.5 0.189 0.192 0.192 0.193 0.192 0.193 0.193 0.193
1 0.255 0.259 0.260 0.259 0.259 0.260 0.260 0.261
2 0.456 0.465 0.466 0.467 0.467 0.468 0.468 0.468
5 1.019 1.064 1.070 1.069 1.069 1.069 1.070 1.070
10 1.870 2.068 2.131 2.131 2.130 2.130 2.129 2.130
20 2.916 3.843 4.131 4.130 4.131 4.131 4.130 4.130
t PPS = 2-phenylphenol, Na salt tetrahydrate.
t Data reported are the mean of three replications using aqueous solutions. Least significant difference - 0.065.
 RHINE ET AL.: IMPROVING THE BERTHELOT REACTION FOR DETERMINING AMMONIUM 479

Table 6. Accuracy and precision of NH^-N analyses by the PPS OU

niicroplate method.! ^~~

'-J
25
,n
<iU ; M M M MA
Z
NHr-N determined^ 15
NHr-N 3? 10
concentration Mean SD Z 5
: : 1^ : : i : :
O) 0
- mg L~ -i. -5
0.5 0.57 0.02 V- -10
1.0 0.95 0.04 Z -15
2.0 1.96 0.04 "J -™
5.0 4.93 0.03 LJJ
10.0 10.04 0.14 DC
20.0 20.02 0.14
I
t PPS = 2-phenylphenol, Na salt tetrahydrate. UJ
| Data reported are the mean of 10 replications using aqueous solutions.
SD = standard deviation.
cr
o
with PPS. Tartrate proved to be ineffective for control- cc
cc
ling precipitation of Ca or Mg, even when color develop- tu
10 15 20 25 30 35 40
ment was carried out in the presence of 18 g of tartrate
L"1. The most widely used chelating agent in Berthelot REFERENCE NUMBER FOR
methods, EDTA (Searle, 1984; Mulvaney, 1996), was INTERFERING COMPOUND
Fig. 5. Effects of the organic and inorganic compounds listed in Table
found to strongly decrease absorbance when present at 2 on color development with 2-phenylphenol, Na salt tetrahydrate
0.9 g L"1, which is less than one-tenth the rate specified (PPS) microplate method at 25°C; NHr-N concentration = (A) 0 or
for NH4-N analysis of soil extracts (e.g., Keeney and (B) 10 mg L"1. Least significant difference = 0.130 mg NH4-N L~'.
Nelson, 1982; Nelson, 1983). Likewise, EDTA has been
found to cause a decrease in absorbance with macro- ment, the PPS-nitroprusside reagent must be added be-
or microscale Berthelot methods that use salicylate fore the buffered hypochlorite reagent.
(Crooke and Simpson, 1971; Kempers and Kok, 1989;
Sims et al., 1995). Kempers and Kok (1989) attributed Evaluation of Modified Berthelot Method
this effect of EDTA to reduced Cl availability. Our
work supports their hypothesis, because studies showed The calibration curves obtained by the microscale
that in the presence of EDTA (up to 11 g L'1), available method described are highly linear (R2 > 0.98). Assum-
Cl was reduced by approximately 33%; moreover, ing complete utilization of NH4-N during color develop-
monochloramine formation was completely eliminated ment, the molar absorptivity of the indophenol dye was
with an EDTA concentration as low as 1.8 g L"1. Based determined to be 2.83 X 104 L mol"1 cm"1.
on these results and previous work by Sims et al. (1995), The accuracy and precision of the microplate method
citrate was used as the chelating agent in the PPS meth- were determined from triplicate analyses performed on
ods described. Studies showed that, with the citrate con- standard NH4-N solutions and soil extracts. With 2 to
centration specified in these methods (9 g LT1), the error 20 mg of NH4-N L"1, analyses were accurate to within
in measuring 10 mg of NH4-N L"1 did not exceed 0.8% 2%, and the CV ranged from 0.6 to 2.0% (Table 6).
in the presence of up to 2 g of Ca L"1 or up to 1 g of Quantitative recovery was obtained in measuring
Mg L-1. NH4-N added to extracts from soils with a wide range
The buffered hypochlorite reagent is unstable and of properties (Table 7). The limit of detection was deter-
should be prepared daily. The PPS-nitroprusside re- mined to be equal to 0.17 mg of NH4-N L"1. To improve
agent is stable in the dark, but should be prepared analyses of samples containing low concentrations of
weekly to ensure maximum sensitivity and should be NH4-N by the macro- or microscale methods described,
stored under refrigeration. For proper color develop- the aliquot taken for analysis can be doubled, and cali-
bration should be done using a range of NH4-N stan-
Table 7. Recovery of NH4-N added to 2 M KC1 soil extracts using
dards that closely brackets the samples.
the PPS microplate method.t__________________ Figure 5 summarizes the results obtained when tests
NHr-N determined?
were performed in triplicate to evaluate the microplate
method described for interference by the 38 organic and
Without added 5 inorganic compounds listed in Table 2. A comparison
N With added N Recovery
of Fig. 5 with Fig. 1 and 2 reveals a striking improvement
Soil Mean SD Mean SD Mean SD in specificity for NH4. Relative to previous Berthelot
M-g —————
o/ methods that use phenol or salicylate, interference by
Plainfield 0.31 0.02 5.48 0.12 103 2.3 amino acids was decreased by up to 10-fold. Interference
Cisne 0.51 0.01 5.74 0.14 104 2.7 by other organic N compounds was virtually eliminated.
Harpster 2.37 0.02 7.52 0.14 103 1.6
Drummer 0.93 0.04 5.96 0.11 101 2.7
Houghton 4.80 0.02 9.70 0.07 98 1.4 ACKNOWLEDGMENTS
t PPS = 2-phenylphenol, Na salt tetrahydrate.
$ Ammonium-N analyses were performed in triplicate on 50 u.L of soil Appreciation is expressed to S.A. Khan for assistance in
extract diluted with 50 (xL of 2 A/ KCI containing 0 or 5 |xg of NHr-N. performing diffusions to check for alkaline hydrolysis of or-
SD = standard deviation. ganic N compounds and to Kerri Marsh for analyses of avail-
480 SOIL SCI. SOC. AM. J., VOL. 62, MARCH-APRIL 1998

able Cl. We also thank T.R. Ellsworth and J.W. Stucki for
use of analytical instrumentation.