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Spectrophotometric Determination of Nitrate with a

Single Reagent
a a
Timothy A. Doane & William R. Horwáth
a
Department of Land, Air, and Water Resources, University of California at Davis, Davis,
California, USA

Version of record first published: 25 Oct 2011.

To cite this article: Timothy A. Doane & William R. Horwáth (2003): Spectrophotometric Determination of Nitrate with a
Single Reagent, Analytical Letters, 36:12, 2713-2722

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ANALYTICAL LETTERS
Vol. 36, No. 12, pp. 2713–2722, 2003

SPECTROMETRY

Spectrophotometric Determination of Nitrate

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with a Single Reagent

Timothy A. Doane* and William R. Horwáth*

Department of Land, Air, and Water Resources,

University of California at Davis, Davis,
California, USA

ABSTRACT

A spectrophotometric procedure for determination of nitrate in

water, soil extracts, and a variety of other sample types is described
using one reagent solution which is easily prepared and stored.
Sample and equipment requirements are minimal. Reduced chemical
hazard, simplicity, and versatility represent improvements over
existing methods. Limit of detection is 0.01 mg N mL
1 (0.72 mM
NO
3 ) or less, depending on the matrix.

Key Words: Nitrate; Spectrophotometry; Single reagent.

*Correspondence: Timothy A. Doane and William R. Horwáth, Department

of Land, Air, and Water Resources, University of California at Davis, 1
Shields Ave., Davis, CA 95616, USA; Fax: 1-530-752-1522; E-mail: tadoane@
ucdavis.edu; wrhorwath@ucdavis.edu.

2713

DOI: 10.1081/AL-120024647 0003-2719 (Print); 1532-236X (Online)

Copyright & 2003 by Marcel Dekker, Inc. www.dekker.com
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2714 Doane and Horwáth

INTRODUCTION

Miranda et al.[1] have presented a sensitive, simple, and convenient

procedure for determination of nitrate. Although described for blood
serum samples, the method may be applied to a wide range of sample
types. Vanadium(III) in acid solution is used to reduce nitrate to nitrite.
As it is formed, nitrite is captured by Griess reagents. The method has
been adapted by combining the reagents into one solution and by using
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cuvets rather than microtiter plates. The latter modification, while less
efficient, is better suited to simple equipment, such as portable or simple
laboratory spectrophotometers. The reagent and analysis are simple to
prepare and carry out, and involve less toxicity, procedural, and
equipment considerations compared to other photometric as well as
nonphotometric methods (e.g., reduction with Devarda’s metal and
steam distillation, nitrate-selective electrode, electrochemical detection,
nitration of organic compounds, UV absorption, or reduction by
cadmium, hydrazine, or nitrate reductase followed by Griess reac-
tion).[2–4] While lacking the potential throughput and multi-analyte
capability of autoanalyzers, the procedure presented here is convenient
enough so that several hundred samples may be processed in one day,
with less investment in equipment and less sample requirement, waste
generation, and preparation time. The procedure is equally convenient
for determination of only a few samples, since the reagent may be stored
and used as needed, with no additional preparation required prior to the
time of analysis. With minor modification, the procedure is adaptable to
automation or field work.

PROCEDURE

For many sample matrices and nitrate concentrations, a dilute

reagent is adequate and is prepared as follows. To 50 mL 1 M HCl add
400 mg vanadium(III) chloride (VCl3). Almost all will dissolve with inter-
mittent gentle shaking (about 10 min). To 400 mL water add 200 mg
sulfanilamide and 10 mg N-(1-naphthyl)ethylenediamine dihydrochloride
(NEDD). Using a syringe filter (1 m) to remove undissolved solids, add
the VCl3 solution to the water. For low concentrations of nitrate and
certain matrices (see discussion below) a more concentrated reagent is
advised: instead of 400 mL water, use 100 mL HCl (0.5–1 M). Although
the stronger reagent may be used in any situation, the dilute reagent can
be used when a stronger reagent is not necessary, simply to avoid excess
use of VCl3.
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Determination of Nitrate 2715

The following volumes of sample and reagent can be mixed directly

in semimicro cuvets, or scaled up to suit the cells for the instrument
being used. In the range of 1 to 20 mg mL
1 NO3-N, mix 20 mL sample
with 1000 mL reagent. For 1 to 10 mg N mL
1, use 45 mL sample and
1000 mL reagent. For 1 to 5 mg N mL
1, use 100 mL sample and 1000 mL
reagent. For less than 1 mg N mL
1, use 500 mL sample and 400 mL
reagent. At concentrations greater than 10 or 20 mg N mL
1, macrocuvets
may be used, and the sample to reagent ratio and volumes adjusted
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accordingly. If a certain matrix presents interference at the high sample

to reagent ratio (500:400), this may be overcome by using concentrated
reagent, decreasing the sample to reagent ratio, or both. (The above
ratios are approximate and may be adjusted as needed.) Color develop-
ment at room temperature (20–25 C) slows down after 4–5 h and is max-
imum after 14–16 h. Measurements are taken at 540 nm. Absorbance
declines approximately 5% from maximum after 48 h. Once color devel-
opment slows down, the exact time of measurement is not important. It is
often convenient to prepare a set of samples one day and read absorbance
the following day, especially when analyzing a large number of samples.
Color may be developed in about 2 h, however, at 60 C.
To demonstrate and evaluate the procedure given above, the
following results are presented. A Shimadzu UV/Vis mini 1240 spectro-
photometer was used for all measurements.

LIMITS OF DETECTION AND DETERMINATION

Using the dilute and concentrated reagent, the absorbances of ten

blanks (water) at decreasing sample to reagent ratios were measured after
20 h at room temperature, and a standard deviation obtained for these
measurements. Limits of detection and determination (quantitation)
were estimated[5] and are given in Table 1. Developing the color at
60 C for 2 h changed these values only slightly (0.001–0.002 mg mL
1
higher for 500:400 sample to reagent ratio; 0.003–0.006 mg mL
1 for
other ratios).

KNOWN MATRICES

Nitrate standard curves in two ranges were prepared in various

matrices. The absorbances of these standards were compared to
standards prepared in water to judge whether or not the matrix interfered
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2716 Doane and Horwáth

Table 1. Limits of detection and determination (quantitation) for the

present procedure.

Detection Determination
Sample:reagent limit limit
(mL) (mg N mL
1; mM NO

3) (mg N mL
1; mM NO
3)

With dilute reagent

500:400 0.002; 0.14 0.005; 0.35
100:1000 0.015; 1.1 0.045; 3.2
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45:1000 0.025; 1.8 0.070; 5.0

With stronger reagent
500:400 0.003; 0.21 0.008; 0.57
100:800 0.010; 0.72 0.040; 2.9

with maximum color development. The concentrated reagent was used

for the low range; dilute reagent was used for the high range, except
for the K2SO4 matrix, which required concentrated reagent to yield
absorbances comparable to a water matrix. Standards were analyzed
three separate times. For convenience, all samples were measured at
room temperature after approximately 20 h (overnight). Zero
absorbance was set using the corresponding blank for each matrix.
These results are shown in Tables 2 and 3. A t-test was used to test for
significance between values obtained for each matrix and those obtained
for a water matrix.
In the range of 1 to 5 mg N mL
1, none of the tested matrices
significantly affected maximum absorbance of standards compared to a
water matrix. (The rate of color development, however, was affected.) A
set of standards prepared in any of these matrices may therefore be used
to analyze samples in any other matrix, provided absorbance is read after
maximum color development.
In the range of 0.01 to 0.1 mg N mL
1, KCl, CaCl2, and substitute
ocean water matrices did not affect maximum color development
compared to a water matrix. In order to overcome interference in
the other matrices, the amount of sample relative to reagent was reduced
to a ratio of 100:800, and these matrices tested using 0.1, 0.5, and
1 mg N mL
1 standards. Absorbances comparable to a water matrix
were obtained (Table 4). However, because the sample to reagent ratio
was reduced, the determination limit for these matrices is necessarily
higher, although it remained below 0.05 mg N mL
1 (3.6 mM NO
3 ) in
all cases.
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1
Table 2. Average absorbance (SD) of three separate analyses of standards in the range 1 to 5 mg N mL prepared in different
Determination of Nitrate

matrices. Dilute reagent was used except with K2SO4 matrix, for which concentrated reagent was used.

Matrix

0.1 M Substitute
Standard phosphate 50 mg/mL ocean
(mg N mL
1) Water 0.5 M K2SO4 0.25 M H2SO4 0.1 M CaCl2 buffer, pH 7 Fe(III) 2 M KCl water[6]

1 0.257 (0.011) 0.242 (0.005) 0.231 (0.015) 0.261 (0.022) 0.237 (0.020) 0.236 (0.026) 0.253 (0.012) 0.258 (0.002)
3 0.722 (0.056) 0.751 (0.026) 0.682 (0.058) 0.742 (0.017) 0.738 (0.049) 0.725 (0.031) 0.728 (0.057) 0.773 (0.009)
5 1.139 (0.073) 1.219 (0.027) 1.145 (0.023) 1.151 (0.023) 1.175 (0.074) 1.152 (0.048) 1.144 (0.084) 1.221 (0.022)
Average r2 0.999 1.000 0.997 0.998 0.999 0.998 0.999 0.998
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2717
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2718 Doane and Horwáth

Table 3. Average absorbance (SD) of three separate analyses of standards in the

range 0.01 to 0.1 mg N mL 1 prepared in different matrices. Concentrated reagent
was used.

Matrix

Standard Substitute
(mg N mL
1) Water 2 M KCl 0.1 M CaCl2 ocean water[6]

0.01 0.017 (0.002) 0.017 (0.001) 0.017 (0.001) 0.017 (0.002)

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0.05 0.079 (0.003) 0.078 (0.004) 0.076 (0.002) 0.083 (0.002)

0.1 0.166 (0.008) 0.159 (0.007) 0.153 (0.004) 0.162 (0.001)
Average r2 0.998 1.000 1.000 0.999

Table 4. Average absorbance (SD) of three separate analyses for matrices which
interfered at lowest sample to reagent ratio (500:400, Table 3). Concentrated
reagent and a sample to reagent ratio of 100:800 were used.

Matrix

0.1 M
Standard phosphate 50 mg/mL
(mg N mL
1) Water buffer, pH 7 0.5 M K2SO4 0.25 M H2SO4 Fe(III)

0.1 0.031 (0.004) 0.030 (0.003) 0.033 (0.005) 0.029 (0.004) 0.029 (0.004)
0.5 0.166 (0.015) 0.167 (0.009) 0.174 (0.018) 0.160 (0.006) 0.161 (0.003)
1 0.320 (0.029) 0.330 (0.021) 0.339 (0.031) 0.311 (0.025) 0.319 (0.010)
Average r2 0.999 1.000 0.999 1.000 0.999

Large amounts of phosphate, sulfate, and acetate changed the color

of the reagent from blue to green, but determination of nitrate was not
affected.

RECOVERY OF MEASURED ADDITIONS OF

NITRATE IN UNKNOWN MATRICES

Accurately accounting for a known amount of nitrate added to a

sample indicates a lack of interference of matrix constituents on determi-
nation. Three samples in complex (unknown) matrices were obtained
with concentrations of NO3-N between 0.1 and 1 mg mL
1. To 1 mL of
each of these samples were added 100 mL (large spike) or 50 mL (small
spike) of 10 mg mL
1 NO3-N standard, resulting in a final NO3-N
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Determination of Nitrate 2719

concentration of (1 þ x)/1.1 (large spike) or (0.5 þ x)/1.05 (small spike),

where x is the original concentration of NO3-N in the sample. Similarly,
five samples were obtained with concentrations of NO3-N between 1 and
10 mg mL
1. To 1 mL of each of these samples were added 100 mL (large
spike) or 50 mL (small spike) of 100 mg mL
1 NO3-N standard, resulting in
a final NO3-N concentration of (10 þ x)/1.1 (large spike) or (5 þ x)/1.05
(small spike), where x is the original concentration of NO3-N in the
sample. Three separate replicates for the large and small spikes were
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prepared from each original sample. Nitrate concentrations were determ-

ined using a calibration with standards prepared in water. All measure-
ments were taken at room temperature after approximately 20 h.
Recovery of the addition was calculated as cf /cm, where cf is the measured
final concentration, and cm the calculated final concentration (given
above), of the spiked sample. These results are shown in Table 5.
Organic-rich matrices were preferably analyzed with concentrated
reagent due to lower recovery of added nitrate with dilute reagent. In
addition, the sample to reagent ratio was reduced for the low nitrate
samples (milk, vetch extract, and wine) to further ensure against any
potential matrix interference.

COMPARISON TO CADMIUM REDUCTION

A set of 10 soil extracts in 1 M KCl, ranging from 1–10 mg mL
1

NO3-N, was analyzed by the proposed procedure and by automated
cadmium reduction/Griess reaction (Lachat Instruments, Milwaukee,
WI; EPA method 353.2). Average difference between results was 3.4%.

REPRODUCIBILITY OF ANALYSIS

Two groundwater samples were determined a total of five times on

five different days, by the same analyst using the same equipment and
same standards. A different batch of reagent was used for each analysis.
Standards were included each time the samples were analyzed and used to
calculate sample concentration. Sample 1 averaged 0.54 mg N mL
1 with
a standard deviation of 0.03. Sample 2 averaged 8.07 mg N mL
1 with a
standard deviation of 0.12.
In general, reproducibility of a manual (pipet) procedure should be
limited only by the consistency with which sample and reagent volumes
are dispensed and mixed each time.
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2720

Table 5. Percent recovery of small and large aliquots of nitrate added to samples. Results are the average of three separate spiked
samples. Standard deviations of measurements are given in parentheses.

Sample to Initial NO3-N Recovery of small Recovery of large

Sample Reagent reagent ratio (mL) (mg mL
1) spike (%) spike (%)

Milk seruma Concentrated 100:800 0.15 (0.006) 98.6 (0.7) 96.9 (2.4)
1:100 Vetch residue extractb Concentrated 100:800 0.14 (0.01) 94.3 (3.4) 94.6 (5.9)
White wine Concentrated 120:800 0.97 (0.02) 102.9 (1.0) 106.2 (0.8)
1:200 Compost extractb Concentrated 30:1000 5.74 (0.22) 99.8 (0.3) 95.7 (0.6)
1:500 Corn residue extract Concentrated 45:1000 3.16 (0.27) 106.0 (1.6) 97.4 (1.7)
River water Dilute 45:1000 2.23 (0.18) 99.5 (3.1) 98.7 (2.1)
Eutrophic river water Dilute 45:1000 2.43 (0.05) 96.5 (0.9) 97.5 (1.5)
Urine Concentrated 30:1000 6.93 (0.32) 100.8 (0.9) 95.8 (0.7)
a
Casein removed with acetic acid.
b
Correction made for background absorbance using a reagent prepared without Griess reagents.
Doane and Horwáth
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Determination of Nitrate 2721

NOTES

1. This procedure measures nitrite together with nitrate; if nitrite is

present in significant amounts it may be quantified separately.
(This may be done with the same reagent—HCl, sulfanilamide,
and NED—without VCl3.) If a significant amount of nitrite is
present, the reaction must be allowed to proceed to completion,
since the color reaction with nitrite is complete within minutes,
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and the results will be affected if the conversion of nitrate as well

is not complete.
2. Potential interferences include S-nitrosothiols and certain
arginine derivatives.[1] Other concerns likely include any
oxidizing agent that might consume V(III) before reduction of
nitrate can occur. Potential interferences in the Griess reaction
include azide, ascorbic acid, and thiols.
3. Quantities of chemicals used in reagent preparation need not be
exact.
4. The acid concentration of the reagent may be increased to
accommodate samples with high pH.
5. V(III) is sensitive to air and light; the reagent is stable for at least
several months if purged (e.g., with N2 or He) and refrigerated
after each use; alternatively, freezing the reagent between uses
also considerably prolongs its stability.
6. If sample concentrations are unknown, several representative
samples may be screened by mixing equal parts sample and
reagent; the same is done for several standards. Heating the
samples (e.g., under hot water) will quickly develop the color;
roughly comparing the samples to the standards will indicate
which range is appropriate.

REFERENCES

1. Miranda, K.M.; Espey, D.A.; Wink, D.A. A rapid, simple spectro-

photometric method for simultaneous detection of nitrate and nitrite.
Nitric Oxide: Biol. Chem. 2001, 26 (1), 62–71.
2. Mulvaney, R.L. Nitrogen—inorganic forms. In Methods of Soil
Analysis; Sparks, D.L., Page, A.L., Helmke, P.A., Loeppert, R.H.,
Soltanpour, P.N., Tabatabai, M.A., Johnston, C.T., Sumner, M.E.,
Eds.; ASA/SSSA: Madison, WI, 1996; 1123–1184.
3. Clesceri, L.S.; Greenberg, A.G.; Eaton, A.D., Eds. Standard
Methods for the Examination of Water and Wastewater, 20th Ed.;
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2722 Doane and Horwáth

American Public Health Association: Washington, DC, 1998;

4.114–4.120.
4. Moorcroft, M.J.; Davis, J.; Compton, R.G. Detection and determi-
nation of nitrate and nitrite: a review. Talanta 2001, 54, 785–803.
5. Currie, L.A. Limits for qualitative detection and quantitative
determination. Anal. Chem. 1968, 40 (3), 586–593.
6. Annual Book of ASTM Standards; American Society for Testing and
Materials: Philadelphia, PA, 1976; Part 31, p. 48.
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Received April 17, 2003

Accepted June 6, 2003