A Simple Spectrophotometric Determination of Nitrate in Water,
Resin, and Soil Extracts
J. E. Yang, E. O. Skogley,* B. E. Schaff, and J. J. Kirn
ABSTRACT
Nitrate is one of the most important chemicals in agricultural and
environmental management. Most methods for NO., analysis, how-
ever, require expensive equipment or complicated procedures. The
objective of this study was to develop a simple, rapid, accurate proce-
dure for NO, analysis that can be conducted in laboratories worldwide,
without the need for specialized equipment. We based our studies on
a colorimetric method that involves electrophilic aromatic substitution
(nitration) between nitronium and salicylate. The simplified procedure
was tested for NO, analysis of water, soil, and resin extracts. Only
1 mL of sample containing 0 to 4 mg NO3-N L"1 is required, and a
single solution (TRI solution made as a mixture of sodium salicylate
[2-hydroxybenzoic acid monosodium salt], NaCl, and ammonium sul-
famate [sulfamic acid monoanunonium salt]) is used for color develop-
ment. The detection limit is 0.1 (ig NO,-N, with linearity up to 4 jxg
NO,-N in a final volume of 11 mL. Evaporation of the sample may
be done in several ways to remove water from reactants, as long as
the temperature for activiation of nitration is attained. Resin extracts
from 2 M HC1 required neutralization with 2 M NaOH prior to
analysis, and soil extracts from 1M KC1 provided more uniform results
than from 2 M KC1. Results from all types of solution samples were
highly significantly correlated with those by the automated Cd-reduc-
tion method. This simplified sodium salicylate procedure (SSP) for
NO, analysis is simple, reproducible, and it requires only inexpen-
sive equipment.
N ITROGEN is THE PRIMARY NUTRIENT most in demand
by plants and microorganisms in the soil. Proper
N management is critical for maintaining productivity
J.E. Yang and J.J. Kim, Div. of Biology and Environment, Kangwon
Natl. Univ., Chuncheon, Korea 200-701; E.G. Skogley and B.E. Schaff,
Dep. of Plant, Soil, and Environmental Science, Montana State Univ.,
Bozeman, MT 59717-0312. Contribution no. J5109 from Montana
Agric. Exp. Stn. and Kangwon Natl. Univ. Received 6 Jan. 1997.
* Corresponding author (eskogley@montana.edu).
Published in Soil Sci. Soc. Am. J. 62:1108-1115 (1998).
and environmental quality. The N cycle is highly dy-
namic, with microbial conversions that may result in
several N forms. Nitrate is the quasi end point of the
cycle, and the form most important in agricultural and
environmental management. Nitrate is the form most
used by plants, it is susceptible to leaching, and it may
undergo several reactions that alter N activity. Nitrates
have been implicated in water pollution (eutrophica-
tion), health problems (infant and animal methemoglo-
binemia), and the formation of carcinogenic nitrosa-
mines. Nitrate management has global significance,
making it important to have available a simple, accurate
procedure for NO3 analysis, especially for laboratories
with limited equipment and resources.
Several methods are in use for NO3 analysis of water
and soil extracts. These include ion chromatography,
flow injection (automated Cd reduction using an auto
analyzer), Kjeldahl distillation, NO3 selective electrode,
and colorimetries such as chromotropic acid, phenoldi-
sulfonic acid, and Brucine (Keeney and Nelson, 1982;
Clesceri et al., 1989). Colorimetries have been widely
accepted for inorganic N analyses because of their sensi-
tivity, rapidity, and ease of use. Some methods are used
mostly for screening purposes, while others are more
versatile but tedious, time consuming, subject to inter-
ferences, and relatively complex. Laboratories that do
not have modern instruments, such as ion chromatogra-
phy and flow- injection systems, often rely on less costly
methods involving colorimetry.
Salicylate has been used for NO3 analyses in water,
soil, and plant extracts with various modifications (Ca-
taldo et al., 1975; Kanno et al., 1968; Scheiner, 1974;
Pharmaceutical Society of Japan, 1990; Korea Standard
Abbreviations: SSP, simplified sodium salicylate procedure; %T, per-
centage of transmittance.
 YANG ET AL.: SIMPLE SPECTROPHOTOMETRIC DETERMINATION OF NITRATE
Methods, 1992). The salicylate method is based on the
electrophilic aromatic substitution reaction, in which
salicylate reacts with nitronium ions (NO?) to form
mostly the nitrobenzoic compound. This converts to a
quinoid compound under alkaline conditions to yield a
yellow color (Vollhardt, 1987; Olah et al., 1989). Color
intensity is proportional to NO3 concentration.
Nitrogen in NO3, however, has no electrophilic capac-
ity, so it must be activated to nitronium, a strong elec-
trophile, by using concentrated H2SO4 and heat:
HO — N H—OSO3H HO — N + HSO4
H * [1]
4- +/>
HO-N H2O + O=N=O
nitronium ion
[2]
Water must be removed for NO3 to be converted to
N02+ (Vollhardt, 1987; Olah et al., 1989), which then
leads to nitration of the benzene ring. Equilibria in
partly aqueous solutions of HNO3 and H2SO4 cause the
formation of hydroxium and hydrogen sulfate ions, thus
reducing the concentration of nitronium ions. Olah et
al. (1989) reported that the rate of nitration fell off
rapidly in media containing H2SO4 in concentrations
<90%, as would be expected from the decrease in the
concentration of nitronium ions. The reaction is irre-
versible (Vollhardt, 1987), so the higher the tempera-
ture, the faster water is removed from the reactants,
and the faster the reaction proceeds to nitronium ions.
In reported procedures (Kanno et al., 1968; Korea
Standard Method, 1992; Pharmaceutical Soc. Japan,
1990), sample and salicylate mixtures were evaporated
to dryness in a water bath, which concentrates samples
low in NO3 and provides conditions for the nitration
reaction to proceed when concentrated H2SO4 is added.
This process is, however, cumbersome and time consum-
ing and limits the number of samples that can be han-
dled, particularly when large-volume water samples
are involved.
An alternative approach to collecting water or soil
samples is the use of resin capsules. Resin capsules are
used in the phytoavailability soil test, as well as in a
wide variety of in situ studies. Bioavailable nutrients
and other elements are accumulated from soil solution
through diffusion kinetics (Yang et al., 1991; Yang and
Skogley, 1992; Skogley and Dobermann, 1996). Cations
and anions adsorbed on the resin are normally recov-
ered by stripping with HC1 or H2SO4. Soil extraction
for NO3 uses KC1 or Ca(OH)2. Chloride in high concen-
trations could form nitrosylchloride to interfere with
NO3 color development (Sadowski, 1969), while resin
and soil extracts have wide pH ranges, ionic strengths,
and Cl~ concentrations as potentially interfering factors
that may require sample pretreatment prior to analysis.
The objectives of this study were to investigate simpli-
fications, reduce time requirements, and expand our
understanding of the salicylate procedure. We based
our studies on the salicylate method described in the
Korea Standard Methods (1992) and Pharmaceutical
1109
Soc. Japan (1990). Study variables included alternative
heating methods for sample evaporation, useful pH
range, and the range of Cl~ inhibition of nitration. We
wanted to develop a rapid, SSP that could be used in
most laboratories for NO3 analysis of water, soil extracts,
or other solutions, including extracts from resin cap-
sules.
MATERIALS AND METHODS
Reagents
A standard stock NO3 solution was prepared by dissolving
0.7218 g of analytical reagent grade KNO3, after drying at
105°C for 6 h, in 1 L of distilled water. This solution contained
100 mg NO3-N L~'. Sodium salicylate was the nitration agent.
We tested 1 to 3% sodium salicylate solutions and found
that the 1% solution was concentrated enough to provide
stoichiometric nitration of salicylate. The more concentrated
solutions gave the same results, but there was no advantage
to their use. We compared (i) 1.0% sodium salicylate solution
in water; and (ii) TRI solution by dissolving 1 g of sodium
salicylate, 0.2 g of NaCl, and 0.1 g of ammonium sulfamate, in
100 mL of 0.01 M NaOH. Sodium chloride, at some minimum
concentration, masks Cl~ interference and ammonium sul-
famate suppresses NC>2~ interferences (Kanno et al., 1968;
Scheiner, 1974), so if these chemicals can be included in a
mixture with sodium salicylate, the procedure could be simpli-
fied. These two solutions were compared in studies to deter-
mine the effects of pH and Cl~ on color development, using
standard NO3 solutions. The other reagents were H2SO4 and
concentrated NaOH (40 g in 100 mL H2O).
Sodium Salicylate Procedure Development
Using Standard Solutions
In this experiment we studied the effects of the two sodium
salicylate solutions and heating (evaporation) methods, using
known concentrations of NO3 in different backgrounds.
Working standard solutions, ranging from 0 to 6 mg NO3-N
L~', were prepared by diluting aliquots of the stock solution
in five backgrounds to represent water samples, resin extracts
in 2 M HC1 or 1 M H2SO4, and soil extracts in 1 M KC1 or
Ca(OH)2 (0.2 g in 100 mL H2O) (Table 1).
One-milliliter aliquots of standard solutions of different
backgrounds were transferred into tall, flat-bottomed test
tubes (2.1 by 11.4 cm). Standard sample vials can also be
used. Solutions in 2 M HC1 or 1 M H2SO4 backgrounds were
neutralized by adding equal volumes of 2 M NaOH. Then,
0.5 mL of sodium salicylate or TRI solution was added and
swirled to mix. Next, this mixture was heated to dryness by
six different heating methods (Table 1): water bath, microwave
radiation, hot plates with different temperatures, and drying
oven at selected temperatures and time. The basic method
involved evaporation by water bath, so we set this as the
standard for comparison. As soon as the tubes were cooled,
1 mL of concentrated H2SO4 was added along the tube wall
to wet the residues. Residues were dissolved by occasional
swirling. After 5 to 10 min, 5 mL of H2O were added slowly
along the wall and swirled to mix (be sure to mix well before
adding NaOH). After cooling, 5 mL of 40% NaOH were
added slowly and mixed well, during which time a yellow color
developed. Tap water was flushed on the outside of the tube
to cool the mixture and the percentage of transmittance (%T)
was measured in a spectrophotometer (Bausch & Lomb, Spec-
tronic 610,1 cm cell, Fisher Scientific, Pittsburgh, PA) at 410
nm. Color intensity was stable for at least 2 d with the tubes
1110 SOIL SCI. SOC. AM. J., VOL. 62, JULY-AUGUST 1998

Table 1. Descriptions of heating (evaporation) methods for stan- samples with low NO3 were concentrated by evaporation in
dard solutions in five different backgrounds. a drying oven, with or without addition of the sodium salicylate
Solution solution. Concentrated samples were frozen at -20°C until
Treatment background Heating methods chemical analysis. Other water samples were acidified with
W:WB Water (W) boiling water bath (WB)
H2SO4 and stored at 4°C. Results from the SSP were correlated
W:MWR microwave radiation (MWR)§ for 3 to with those from the automated Cd-reduction method, a cur-
4 min rent standard procedure.
W:HP3M hot plate (HP)§ at high temp (450° C) for Resin capsules, which had been used to monitor NO3 move-
3 min ment through soils in the field, were washed and stripped with
W:HP1H hot plate (HP) at low temp (80-130°C) for
Ih 2 MHC1 to recover adsorbed ions (Yang et al, 1991; Yang and
W:OV65ON drying oven (OV) at 65° C for overnight Skogley, 1992; Skogley and Dobermann, 1996). We selected 54
(16 h) samples showing a wide range of NO3 concentrations. Another
W:OV1053H drying oven (OV) at 105° C for 3 h 54 resin capsules, used to monitor Br~ transfer in field experi-
H:WB 2 JWHCI boiling water bath (WB) ments, were stripped with 1 M H2SO4. Resin extracts (1 mL)
H:MWR (H/Resin microwave radiation (MWR) for 3 to in acid backgrounds were neutralized with 1 mL of 2 M NaOH
extracts) 4 min
H:HP3M hot plate (HP) at high temp (450° C) for and analyzed by SSP and automated Cd reduction.
3 min Samples of 31 soils collected from agricultural fields in
H:HP1H hot plate (HP) at low temp (80-130°C) for Gallatin County, Montana, were extracted with 1 M KC1 and
Ih 35 samples were extracted with 0.2% Ca(OH)2 (Haby, 1989).
H:OV65ON drying oven (OV) at 65° C for overnight
All extracts were analyzed by SSP and automated Cd re-
(16 h)
H:OV1053H drying oven (OV) at 105° C for 3 h duction.
H:OV105ON drying oven (OV) at 105° C for overnight
(16 h)
S:WB 1 M H2SO4 boiling water bath (WB) RESULTS AND DISCUSSION
S:MWR (S/Resin microwave radiation (MWR) for 3 to
Nitration Reagent
extracts) 4 min
S:HP3M hot plate (HP) at high temp (450° C) for
3 min Previous procedures (Kanno et al., 1968; Korea Stan-
S:HP1H hot plate (HP) at low temp (80-130°C) for dard Method, 1992; Pharmaceutical Soc. Japan, 1990)
Ih used separate solutions of sodium salicylate, NaCl, and
S:OV65ON drying oven (OV) at 65° C for overnight
(16 h) ammonium sulfamate. To test the need for separate
S:OV1053H drying oven (OV) at 105° C for 3 h solutions, sodium salicylate was prepared as a single
S:OV105ON drying oven (OV) at 105° C for overnight
(16 h) solution or as a TRI solution as described above. Color
intensities of standard NO3 solutions analyzed with the
K:WB 1MKC1 boiling water bath (WB)
K:MWR (K/Soil microwave radiation (MWR) for 3 to TRI solution (Y) were highly correlated with those ana-
extracts) 4 min lyzed with sodium salicylate (X) (Y = 2.19 + l.OLY,
K:HP3M hot plate (HP) at high temp (450° C) for r = 0.999, at P < 0.01). The slope of nearly one indicates
3 min
KrHPlH hot plate (HP) at low temp (80-130°C) that color intensities were essentially equal. Standard
forlh N solutions having a range of NO3 concentrations also
K:OV65ON drying oven (OV) at 65° C for overnight
(16 h) were analyzed by using the TRI solution, rather than
K:OV1053H drying oven (OV) at 105° C for 3 h the separate addition of the three chemicals. Again, the
C:MWR 0.2% microwave radiation (MWR) for 3 to %T yielded a highly significant correlation coefficient
Ca(OH)2 4 min (r) of 0.99 (P < 0.01), with a slope of nearly one. These
C:HP3M (C/Soil hot plate (HP) at high temp (450° C) results indicate no advantage to separate addition of
extracts) for 3 min
the three chemicals.
t For microwave radiation (MWR) and hot plate (HP), one or two pieces Certain minimum concentrations of NaCl have been
of porous boiling carbon chips (Fisher Sci. Co.), washed thoroughly with
H2SO4, were added to prevent abrupt eruption of solution. used to swamp out Cl~ interference, and ammonium
sulfamate has been used to remove NO^ interference
capped. Of several combinations investigated, the ratio of (Kanno et al., 1968; Scheiner, 1974). If solutions do not
concentrated H2SO4/H2O/NaOH = 1:5:5 worked well, so this contain significant concentrations of Cl~ or NO2~, there
protocol was used in all experiments, unless otherwise noted. is no need to add chemicals to eliminate interference.
Each chemical was added by preset dispensers. Screening for the presence of these ions, however, re-
Total final volumes were adjusted to 11 to 50 mL, with quires separate, complicated procedures (Keeney and
water, to test linearity. Standard solutions of 2 mg NO3-N LT1 Nelson, 1982) that are much more time consuming than
were prepared in pHs ranging from 1 to 10, using dilute H2SO4 that required to make and use the TRI solution. Because
or NaOH, to determine the useful pH range. Standard solu- results are not significantly different, we recommend
tions having 2 mg NO3-N L"1 were prepared in solutions
having Cl~ ionic strengths ranging from 0 to 2 M with NaCl. using the TRI solution routinely in the SSP to protect
against potential interferences.
Sodium Salicylate Procedure Calibration
with Field Samples Detection Limits of the Sodium
Salicylate Procedure
Ten water samples were collected from wells, groundwater,
irrigation ditches, and freshwater creeks in the Gallatin Valley, Figure 1 shows the calibration curve of the SSP for
Montana. These samples were analyzed immediately by the standard solutions in water, 2 M HC1 (resin extract), or
SSP and by automated Cd reduction for comparison. Water 1 M KC1 (soil extract) backgrounds. Regression equa-
 YANG ET AL.: SIMPLE SPECTROPHOTOMETRIC DETERMINATION OF NITRATE 1111

Table 2. Linear regression equations between percent transmit-

tance (Y) and NO3-N concentrations (A')t for the standard
solutions of water, resin, and soil extracts evaporated by differ-
ent methods.
Treatment Regression equations r2§
0)
u W:WB Y = 97.56 - 12.76* 0.986**
c W-.MWR Y = 97.61 - 12.51* 0.988**
re
W:HP3M Y = 97.30 - 12.17* 0.986**
W:HP1H Y = 97.46 - 13.07* 0.984**
E W:OV65ON Y = 97.58 - 12.76* 0.989**
Cfl
c W:OV1053H Y = 97.85 - 12.91* 0.991**
re
H:WB Y = 98.82 - 10.09* 0.996**
H:MWR Y = 100.20 - 8.60* 0.999**
H:HP3M Y = 100.00 - 8.25* 0.999**
H:HP1H Y = 99.48- 10.81* 0.997**
H:OV65ON Y = 97.17 - 5.43* 0.886*
H:OV1053H Y = 97.63 - 11.14* 0.993**
H:OV105ON Y = 99.05 - 10.46* 0.988**
NO3-N, ug S:WB Y = 99.63 - 10.72* 0.999**
Fig. 1. Calibration curve of the simplified sodium salicylate procedure
S:MWR y = 98.42 - 12.20* 0.995**
(SSP) for standard NO., solutions in water, 2 M HCI, and 1 M
S:HP3M y = 99.28 - 10.65* 0.999**
S:HP1H Y = 97.65 - 11.56* 0.988**
KC1 backgrounds. S-.OV65ON Y = 99.90 - 10.93* 0.999**
S:OV1053H Y = 99.76 - 10.51* 0.998**
S:OV105ON Y = 99.57 - 11.27 0.997**
tions and r values for these curves for the 0- to 4-jjig
K:WB Y = 99.02 - 11.12* 0.997**
range are as follows: K:MWR Y = 97.97 - 9.42* 0.999**
K:HP3M Y = 98.99 - 8.99* 0.997**
water background: Y(%T) = 98.98 - 1435X K:HP1H y — 98.78 - 10.67* 0.991**
(NOy-N, fig); r = -0.9995 (P < 0.001) K:OV650ON ¥f
99.38
__
- 10.93* 0.998**
K:OV1053H K = 99.62 - 10.64* 0.999**
2 M HCI background: Y(%T) = 100.02 - 8A1X C:MWR Y = 98.30 - 12.69* 0.993**
(NO3-N, |xg); r = -0.9997 (P < 0.001) OHP3M Y = 97.39 - 12.12* 0.975**
1 M KC1 background: Y(%T) = 99.73 - 10A6X t NO3-N concentration ranged from 0 to 4 g mL '.
(NOj-N, ug); r = -0.9999 (P < 0.001) $ Refer to Table 1 for descriptions.
§ r2 values significant at P values <0.01 (**) and <0.05 (*) are 0.919 and
Equations for these curves and for the different heating 0.771, respectively.
methods are listed in Table 2. A final volume of 11 mL
yielded a highly significant linear relationship between with 2 M KC1 or with 1M KC1; therefore, we recommend
the %T and NO3-N up to 4 u,g. Above this level the 1 M KC1 or 0.2% Ca(OH)2 for soil extraction when the
curve deviated from linearity; this also occurred for all SSP is to be used.
solution volumes at NO3-N levels >6 |xg. The lower Linearity could be extended up to =16 (Jig NO3-N,
detection limit was 0.1 to 0.2 u.g NO3-N with %T read- after color development, by dilution with water to a
ings of 96 to 97. final volume of 50 mL, as depicted in Fig. 2. Highly
The low pH and high Cl~ concentrations of resin and significant linear correlations with r values >0.996 (P <
soil extracts interfered with color development, as indi- 0.001) existed for the linear portions of the curves. The
cated in the lower slopes of the curve (Table 2). Strong extent of linearity depended on the final volume of the
interference occurred in the 2 M HCI resin extracts. color developing solution. These results illustrate that
Neutralization of resin extracts with 2 M NaOH resulted
in highly significant linearity (Fig. 1, Table 2). Similarly,
neutralization of 1 M H2SO4 resin extracts gave highly
significant linearity between 0 and 4 u.g NO3-N. Sulfate
ions did not appear to interfere as much as Cl ions. If
40% NaOH was added as the final step, rather than o>
prior to analysis, color did not develop normally. This o
c
indicates interferences were due to low pH, high Cl~
concentration, or both, and that neutralization of resin fi
extracts is required prior to evaporation and analysis 1
(0
by this method. Using 2 M NaOH for neutralization re
allows pH adjustment without excessive sample dilution.
Calibration curves for 2 M KC1 soil extracts could
not be made, apparently due to Cl" interference. The
1 M KC1 and 0.2% Ca(OH)2 extracts, however, allowed
normal color development with highly significant linear-
ity (Table 2). Results from the Montana State University N03-N (ug)
Soil and Water Analytical Laboratory (1997, unpub- Fig. 2. Linearity of NO., analyses by the simplified sodium salicylate
lished data) indicate no difference in soil NO3 extraction procedure (SSP) at different final volume dilutions with water.
1112 SOIL SCI. SOC. AM. J., VOL. 62, JULY-AUGUST 1998

Table 3. Correlations (percentage of transmittance) between dif- nearly one. Pearson correlation coefficients (Table 4)
ferent evaporation methods and the water bath (WB) method
for the standard solutions! having the same background.
verify the highly significant relationships between heat-
ing methods for various background solutions.
Regression Evaporating mixtures of 1 mL of sample and 0.5 mL
model!
Background of TRI solution to dryness in a microwave oven (600 W)
solution Y X Regression equations r2 or on a hot plate (Corning Hot Plate PC-351, Corning,
Water MWR WB Y = 2.03 + 0.98* 0.999** NY; high setting =450°C) took =2 to 3 min. Solutions
HP3M WB ¥ = 4.24 + 0.95* 0.999** tended to erupt or sputter due to rapid heating unless
HP1H WB Y = -2.54 + 1.03* 0.999**
OV65ON WB Y = 0.15 + 1.00* 0.999** one or two pieces of porous carbon boiling chips (Fisher
OV10S3H WB Y = -0.67 + 1.01* 0.999** Scientific, Pittsburgh, PA) were added. Boiling chips
2MKC1 MWR WB Y = 16.36 + 0.85* 0.994*** were pretreated by thoroughly washing with water and
(Resin HP3M WB F = 19.62 + 0.8LY 0.993** concentrated H2SO4. No contamination or interference
extracts) HP1H WB F = -6.33 + 1.07* 0.999**
OV65ON WB Y = 43.14 + 0.55* 0.923** from the boiling chips could be detected. Precaution
OV1053H WB Y = -9.47 + 1.10* 0.997** should be taken, however, to remove samples from heat
1 M H2SO4 MWR WB Y = -15.00 + 1.13* 0.997*** before a burnt or brownish color occurs in the dried
(Resin HP3M WB Y = 0.31 + 0.99* 0.992** salts. Such charring often interfered with normal
extracts) HP1H WB Y = -9.94 + 1.08* 0.992**
OV65ON WB Y = -1.62 + 1.02 * 0.999** color development.
OV1053H WB Y = 2.10 + 0.98 * 0.999** Exposing sample and TRI solution mixtures to high
1MKC1 MWR WB F = 16.31 + 0.84* 0.997*** temperatures for 30 sec to 1.5 min in the microwave
HP3M WB y = 19.00 + 0.81* 0.998** oven or on the hot plate, but without complete drying,
HP1H WB Y = 3.63 + 0.96* 0.998** did not allow normal color development. If samples
OV6SON WB Y = 2.12 + 0.98* 0.999**
OV1053H WB Y = 5.01 +ms 0.96* 0.999** were nearly dry, adding more concentrated H2SO4 al-
t NOj-N concentration ranged from 0 to 4 g mL~'.
lowed full color development. This in turn required
I Refer to Table 1 for descriptions. more NaOH for color development. Olah et al. (1989)
** Significant at P < 0.01. showed the rate of nitration was 100% at H2SO4 concen-
tration of 96.7% but dropped off at 91.2%, supporting
samples with high NO3 levels could be directly analyzed the use of concentrated H2SO4 to ensure complete ni-
without dilution of the original sample. tration.
From these results, we suggest maintaining 0 to 4 (jug It is clear that several methods will serve to remove
NO3-N concentrations in standard solutions and the water from the samples, and that the duration of heating
analyte for analysis of water, resin, and soil extracts by is not important as long as water is completely removed
the SSP, using a final volume of 11 mL. and the activation temperature is attained. Researchers
may select any appropriate heating method for their
Heating Methods experimental purposes and convenience.
Removal of water from reactants is critical to at-
taining the temperature for activation of NO3 and sub- pH
sequent nitration reactions of electrophilic aromatic Figure 3 shows the effects of pH between 1.06 and
substitution (Olah et al., 1989; Vollhardt, 1987). We 10.1 on color development in samples containing 2 mg
compared six heating and evaporation methods (Table NO3-N L"1 in water background. Samples were mixed
1), with the water bath used as the standard. Table 3 with either sodium salicylate or TRI solution and pHs
shows highly significant correlations between all heating were adjusted using dilute H2SO4 or NaOH.
methods and the water bath for analysis of standard The critical pH of the SSP was 2, below which color
solutions (0-4 jxg NO3-N) in water, resin, and soil ex- development was strongly inhibited. Color development
tracts. In most cases, slopes of these relationships were was the same as for standards at all pHs >2, with no
Table 4. Pearson correlation coefficients (r)f between evaporation methods^ of standard solutionsg having different backgrounds.
Water HO (Resin extract) KC1 (Soil extract)
WB MWR HP3M OV1053H WB MWR HP3M OV1053H WB MWR HP3M OV1053H
Water WB
MWR 0.9999
HP3M 0.9998 0.9999
OV1053H 0.9997 0.9999 0.9997
HC1 WB 0.9985 0.9991 0.9986 0.9995
MWR 0.9915 0.9928 0.9916 0.9942 0.9970
HP3M 0.9900 0.9916 0.9906 0.9931 0.9962 0.9997
OV1053H 0.9978 0.9978 0.9969 0.9983 0.9986 0.9957 0.9939
KC1 WB 0.9981 0.9987 0.9981 0.9993 0.9999 0.9975 0.9967 0.9990
MWR 0.9930 0.9943 0.9933 0.9956 0.9978 0.9995 0.9993 0.9966 0.9983
HP3M 0.9965 0.9975 0.9971 0.9983 0.9994 0.9980 0.9980 0.9970 0.9994 0.9989
OV1053H 0.9960 0.9968 0.9960 0.9977 0.9993 0.9991 0.9984 0.9984 0.9996 0.9995 0.9993
t The r values required to be significant at I' 0.01 are 0.959.
t Refer to Table 1 for the method abbreviations,
§ NOj-N standard solutions ranged from 0 to 4 jig mL~'.
 YANG ET AL.: SIMPLE SPECTROPHOTOMETRIC DETERMINATION OF NITRATE 1113

120

100

80 - sfd 2 ug
or*
-e—o-o o
0) 60 -
•-• T % (sample + Na-Salicylate in water)
C O-O T % (sample + Na-Salicylate in NaOH)
2 20
HI pH (sample + Na-Salicylate in water)
I I pH (sample + Na-Salicylate in NaOH)

E
</> 15
c pH after mixing
(0

0 Illllll
pH of Sample Solution
Fig. 3. Effects of initial sample pH of solutions containing 2 p-g NO,-N on the percentage of transmittance (line), and pH of solutions after
mixing with sodium salicylate (bar).

differences between samples mixed with sodium salicy- Cl from samples having excessively high concentra-
late or TRI solution. The TRI solution had a pH of 9.8 tions by treatment with AgSO4.
and sodium salicylate was pH 7.2. When these reagents
were added to samples, pHs increased as shown in Fig. Recommended Procedure
3. Samples mixed with TRI solution were buffered at
pH 9.6 for samples having an initial pH >3, but if the Based on results of this study, we recommend the
initial pH was <2, mixture pHs were 3.3 or lower. Sam- following standard procedure as a simplified version of
ples mixed with sodium salicylate had lower pHs than the salicylate (SSP) method for NO3 analysis:
those of TRI solution, but these pH differences did 1. Transfer 1 mL of sample containing 0 to 4 mg
not influence color development. These results provide NO3-N L"1 into a flat-bottom sample vial.
further evidence that the TRI solution may be success- 2. Add 0.5 mL of TRI solution.
fully used for a broad range of samples, with no pH 3. Evaporate to dryness on a hot plate (or in an oven
adjustment required for water or soil extracts prior to overnight) and cool the residue.
analysis. Resin extracts from strong acids will, however, 4. Wet the residue with 1 mL of concentrated H2SO4
require neutralization prior to analysis by SSP. and allow to stand for 5 min.
5. Add 5 mL of H2O down the vial wall and swirl to
Chloride Molarity mix. Allow the solution to cool.
Figure 4 shows the effects of Cl~ molarity on color
development. Standard solutions (2 mg NO3-N L"1),
prepared in TRI solution or sodium salicylate, showed
no significant difference in the %T at all levels of addi-
tional Cl~. Concentrations of Cl~ above 0.1 M, however,
reduced color intensity with both nitration agents, and 5%
above 0.2 M, strong Cl~ interferences occurred in pro- std
portion to increasing concentration. Scheiner (1974) re-
ported negligible interference from 1500 mg Cl' L"1 i
C 60 H
(=0.04 M) in a similar procedure. Sadowski (1969) re-
c
ported that high concentrations of Cl~ might react with flj 50
NO3 to form nitrosylchloride, thus reducing the concen-
tration of nitronium ions for the nitration of salicylate.
Standard curves remained linear in a background of
1 M KC1, but not in 2 M KC1 (Table 3). Based on these
results, we recommend that NO3 extraction of soils for
analysis by SSP be done with the less concentrated ex- Cl" concentrations (mole/L)
tracting solution, and that the TRI solution should con- Fig. 4. Effects of Cl molarity on the percentage of transmittance of
tain 0.03 to 0.05 M Cl~. It may be necessary to remove solutions containing 2 (xg NO3-N.
1114 SOIL SCI. SOC. AM. J., VOL. 62, JULY-AUGUST 1998

6. Add 5 mL of 40% NaOH down the vial wall, swirl,
and cool.
7. Read absorbance or transmittance at 410 nm.
8. Calculate NO3-N concentration from the standard
calibration curve.
Calibration of Sodium Salicylate Procedure
with Cadmium Reduction on Field Samples
Water samples, soil extracts, and resin-stripping solu-
tions from various field sites and experiments were ana-
lyzed by the SSP and compared with results of analysis
by automated Cd reduction, which we used as the cur-
rent standard method. Figure 5 presents results for wa-
ter, resin extracts, and soil extracts. Correlations be-
tween the two methods were highly significant for all
samples, illustrating that the SSP is appropriate for NO3
analysis of various types of samples.
We also concentrated six water samples, with or with-
out the addition of sodium salicylate, and analyzed for
NO3 by the SSP. The equation for correlation between
these two methods of concentrating the water was Y =
0.004 + 1.042^, r = 0.99***, indicating no loss of NO3
during evaporation when sodium salicylate was not
added. Water samples with low concentrations of target
elements, such as N, P, K, or metals, often must be
concentrated prior to analysis. Evaporation of large vol-
umes containing sodium salicylate is very slow and cum-

(a)
O)
3Y = 0.015 + 1.007X, r = 0.99*** (n = 54)
£ 4 = -O.*f + LOfX, r = 0.99*** fn = 10)
O
3
1
•o
£ 2
•O
1
U
A

1 2 3 4 5
1 2 3 4 NO3 -N by SSP Method (mg/L)
-N by SSP Method (mg/L)

~. 6
Y = 0.002 + 0.82X, r = 0.98*** (n = 31)
Y = 0.14 + 0.94X, r = 0.97*** (r = 54;

I 2 3 4 5 1 2 3 4

NO3 -N by SSP Method (mg/L) N03-N by SSP Method (mg/L)

2 » Y = 0.01 + 0.82X, r = 0.98*** (n = 35;

-N by SSP Method (mg/L)

Fig. 5. Correlation between sodium salicylate (SSP) and automated Cd-reduction methods for samples of (a) water, resin extracts in (b) 2 M
HCl or (c) 1 M H2SO4, and soil extracts in (d) 1 M KC1 or (e) 0.2% Ca(OH)2.
 YANG ET AL.: SIMPLE SPECTROPHOTOMETRIC DETERMINATION OF NITRATE 1115

bersome, and problems in color development were re-
ported due to oxidation of organic matter when
concentrated H2SO4 was added to the dried residue. To
circumvent these problems, we suggest evaporating an
appropriate volume of sample to within the analytical
range and using a 1-mL aliquot of the concentrate for
NO3 analysis by SSP.
adopted for use in laboratories with limited equipment
and resources.
ACKNOWLEDGMENTS
This research is supported in part by the Korea Ministry
of Education Research Fund and Agricultural Experimental
Station, Montana State University, Bozeman.

SUMMARY AND CONCLUSIONS

A number of methodologies are used to analyze NO3
in samples of environmental importance; however, most
current procedures are either slow and cumbersome or
require expensive equipment. We wanted to develop a
rapid method that could be used in laboratories with
limited equipment or resources, while maintaining sen-
sitivity and accuracy. Based on a procedure that uses
sodium salicylate as a nitration agent to develop a yellow
color, several parameters were compared that might
allow simplification of the reported procedure, while
also decreasing the time required for analysis.
Results of this study indicate that a simplified sodium
salicylate procedure can be successfully used to accu-
rately determine NO3 concentrations in water samples,
salt extracts from soils, or acid extracts from resin cap-
sules. Simplifications include using a hot plate, micro-
wave, or convection oven to concentrate and dehydrate
samples; combining three chemicals in the nitration
agent to eliminate potential unknown interferences; and
neutralization of strongly acid extracts prior to analysis.
An appropriate aliquot of samples having low NOs con-
centrations can be evaporated to obtain the desired
concentration range, and then only 1 mL of this concen-
trated sample is needed for analysis. The detection limit
is about 0.1 fig NO3-N and results correlated strongly
with those from the automated Cd-reduction method.
Using the SSP will allow analysis of large numbers of
samples in a short time, and the procedure can be