Nitric Oxide 57 (2016) 79e84

Contents lists available at ScienceDirect

Nitric Oxide
journal homepage: www.elsevier.com/locate/yniox

A new, fast and sensitive method for the determination of trace

amounts of nitrite using differential pulse polarography
Gler Somer*, Skr Kalayc, Ziya Almas
Gazi University, Chemistry Department, 06500, Ankara, Turkey

a r t i c l e i n f o a b s t r a c t

Article history: Nitrite salts of sodium or potassium are being used for the protection of meat products. They provide
Received 1 February 2016 color and taste of meat and they protect against clostridia. On the other hand, nitrite ions can interact
Received in revised form with amines to form nitrosamines which are known as carcinogenic substances. They may also react with
15 April 2016
hemoglobin and reduce its oxygen carrying capacity. Thus, its concentration in food-stuff has to be
Accepted 2 May 2016
controlled carefully by highly sensitive methods.
Available online 3 May 2016
A new DP polarographic method is established for the determination of nitrite. Nitrite cannot be
determined directly with any analytical methods. Long and tedious procedures are necessary for many of
Keywords:
Nitrite
them. In this polarographic method arsenite, As(III), ion is used for the reduction of nitrite. The nitrite is
Trace determination determined from the As(III) quantity left over after the reaction with nitrite. The peak of arsenite has
DP polarography been used since it is sharp and responds well for the standard addition of arsenite.
Interference studies The optimum conditions for the quantitative reaction between nitrite and arsenite have been studied.
It was found that the pH for the reaction medium has to be 5e7, since nitrite is decomposed at lower pH
values. The reaction medium has to be stirred for about 5 min with nitrogen gas in order to expel the NO
gas formed and thus to shift the equilibrium to products side. The limit of detection, LOD, was found to be
as 2
107 M. No interference was observed from most common ions.
2016 Elsevier Inc. All rights reserved.

1. Introduction
The food-stuff protection has taken an important place in in-
dustry. Nitrite salts of sodium or potassium are being used for the
protection of meat products. They provide color and taste of meat
and they protect against clostridia. On the other hand, nitrite ions
can interact with amines to form nitrosamines which are known as
carcinogenic substances. They also may react with hemoglobin and
reduce its oxygen carrying capacity [1,2].
Thus, its concentration in food-stuff has to be controlled care-
fully by highly sensitive methods. The commonly used methods for
nitrite determination include the spectrophotometric [3e6], chro-
matographic [7,8], FIA-CL ow injection-chemiluminescence [9,10]
and electrochemical methods such as amperometry [11], voltam-
metry [12e16], and polarography [17e20].
Nitrate and nitrite in atmospheric liquids and lake waters could
be determined by reverse-phase high performance liquid
* Corresponding author.
E-mail addresses: gsomer@gazi.edu.tr (G. Somer), skalayci@gazi.edu.tr
(S. Kalayc), zalmas@gazi.edu.tr (Z. Almas).
chromatography [7]. Nitrite and nitrate ions were determined after
reduction with NaI to NO and the chemiluminescence of NO in the
presence of ozone was measured [9].
The amperometric determination of both nitrite and nitrate was
made using tetraruthenated porphirin-modied electrode. This
method was applied for nitrite and nitrate determination in min-
eral water and in cured meat [11]. An anodic stripping procedure
was described for nitrite using cetyltrimethylammonium bromide
as a complexing agent. The oxidation of this complex on a glassy
carbon electrode was used for nitrite determination [12].
Electrochemical sensors using modied electrodes based on
copper complexes were used for determination of nitrite in water
[13]. In one work nitrate and nitrite was determined using DPP
(differential pulse polarography). The reduction peak of nitrite in
the presence of Zr (IV), which acts as a catalyst, was used for the
destruction of nitrite [17]. By using the selective reduction of both
nitrate and nitrite into nitric oxide both ions could be determined
by DPP [18]. In another work o-,m- or p-aminobenzoic acids were
used for DPP-determination of nitrite from the decrease of their
peak by the addition of nitrite [19]. In our former studies nitrite was
determined using DP polarography by direct and indirect methods.

http://dx.doi.org/10.1016/j.niox.2016.05.001
1089-8603/ 2016 Elsevier Inc. All rights reserved.
80 G. Somer et al. / Nitric Oxide 57 (2016) 79e84
Sulte ion was used for the reduction of nitrite for the indirect
determination [20].
The classical colorimetric methods widely used, are based on
the formation of strongly colored azo dyes. For this purpose an
amine is diazotized by means of nitrite and then the intermediate is
let to couple with a selected aromatic compound. Most of the above
mentioned methods are laborious, since in many of them diazoti-
zation and coupling reactions are used; some of them suffer from
matrix interference by oxidizing and reducing agents.
Electrochemical methods offer useful alternatives since they
allow faster and cheaper analysis. The results obtained with DPP
method are very reproducible since with the use of dropping
mercury electrode (DME) the behavior of the electrode is inde-
pendent of its past history.
In this work, a new indirect polarographic method was devel-
oped where it was possible to determine very low concentrations of
nitrite using arsenite As(III) ion for the reduction of nitrite. For this
purpose as given below the electrochemical behavior of nitrite and
As(III) have been studied and the optimum conditions for the
quantitative reaction conditions have been determined. This
method can be applied to many biological samples safely.
2. Experimental
2.1. Apparatus
A polarographic analyzer Entek Electronics Model 2016
equipped with a PAR mercury drop timer was used. The drop time
of the electrode was in the range 2e3 s (2.35 mg s1). A Kalousek
electrolytic cell with a saturated calomel electrode (SCE), separated
by a liquid junction, was used in the three-electrode conguration.
With this three eelectrode cell the danger of IR drop (where
I current, and R resistance) is prevented. All potentials in the
manuscript are given vs. SCE. The counter electrode was platinum
wire. The polarograms were recorded with a Linseis (LY 1600) XeY
recorder under the conditions of a drop life of 1 s, a scan rate of
2e5 mV s1, and a pulse amplitude of 50 mV.
2.2. Reagents
All chemicals used were of analytical-reagent grade (proanal-
ysis). Triply distilled water was used in the preparation of all so-
lutions. In order to expel the oxygen present in polarographic cell
99.999% pure nitrogen was passed through the solution. Solutions
of 1
103 M and more diluted ones were prepared before every
use in order to avoid the aging process of solution.
The mercury used in the dropping mercury electrode was ob-
tained from Merck (Darmstadt, Germany). Contaminated mercury
was cleaned by passing it successively through dilute HNO3 (3.0 M)
and water columns in the form of ne droplets by using a platinum
sieve. This mercury is then washed in the same way until no acidic
reaction was observed. The collected mercury was stored in a
closed vessel covered with water. It was dried between sheets of
lter paper when it was needed. The mercury used is not lost since
it was collected quantitatively, without causing any pollution. Thus,
no mercury loss is possible and it can be used continuously. Before
use, a DPP polarogram of this mercury was recorded in order to
conrm the absence of impurities.
2.3. Preparation of reagents
2.3.1. 1.0 M AcOH/AcO electrolyte
It was prepared by adding 6 g of solid NaOH, which had to be
washed rst with distilled water to remove the carbonate formed,
into 57 mL of 1.0 M AcOH and diluting to 1 L. The pH was adjusted to
the desired value using a pH meter.
2.3.2. 0.1 M KNO2 solution
Dried 0.21 g of KNO2 was dissolved in 25 mL de-aerated distilled
water. It was stored in dark.
2.3.3. 0.1 M As(III) solution
About 0.65 g NaAsO2 was dissolved in distilled water and diluted
into 50 mL using volumetric ask.
1.1 M Pb(II), Zn(II), and Cu(II) solutions were prepared from their
standard nitrate solutions; only for Cd(II) solution the chloride salt
was used.
3. Results and discussion
3.1. Electrochemical behavior of nitrite ion
In DP polarography the nature (cathodic or anodic) of a polar-
ographic peak cannot be evaluated. For this purpose direct current
(DC) polarography has to be used. In our former studies with DC, we
have shown that 1
104 M nitrite ion is reduced in several
electrolytes and it showed a cathodic current on DME at
about 1.43 V.
The DP polarograms of nitrite were taken in varying concen-
trations of HCl (6, 4, 2, 1 and 0.1 M) and in acetate buffers. It was
observed that in these acidic solutions nitrite was decomposing and
NO gas was escaping. Thus, it is not possible to determine nitrite
concentration correctly. It was found that in 4 M HCl medium while
nitrogen gas was purging from 2
104 M nitrite 10% was
decomposed in 8 min, in 6 M HCl on the other hand 20% of nitrite
was decomposed in 4 min, Fig. 1. However, it was found that
1
104 M nitrite peak did not decrease with time at about pH 5
HAc. Table 2.
3.2. Indirect determination of nitrite using arsenite As (III)
According to our former work it is decided that lower concen-
trations of nitrite cannot be determined precisely since nitrite peak
is not high enough. Thus, it will be better to nd a substance which
is electroactive and reacts with nitrite quantitatively. As can be seen
from the reduction potentials given below, arsenite ion possesses
these feature, it can react with nitrite ion quantitatively.
 G. Somer et al. / Nitric Oxide 57 (2016) 79e84 81

Table 2
Dependence of acidity on decomposition of nitrite.

Acid molarity Time Decomposition

4 M HCl 8 min 10%

6 M HCl 4 min 20%
HAc pH 5 e No decomposition
HAc pH 7 e No decomposition

about 0.85 V belongs to the reduction of As into Arsenide, As (3).

As can be seen As (III) is electroactive, its peak is much higher
than nitrite peak. Thus, it can be used for the indirect determination
of small concentrations of nitrite safely.

3.4. Optimum reaction conditions

The reaction between As (III) and nitrite may be quantitative

only under optimum conditions. The formation of NO in gaseous
state gives an advantage since it will shift the equilibrium to
products side, during purging nitrogen gas through the reaction
medium. Here the acidity is also important since nitrite itself may
decompose before it reacts with As (III). After careful examination
for a quantitative reaction, the optimum conditions in reaction cell
could be designated as:

- the pH for the reaction medium has to be 5e7, since at lower pH

values nitrite itself is decomposed,
- it has to be stirred for about 5 min with nitrogen gas in order to
expel NO gas formed,
- As (III) concentration has to be at least 4 times or larger of nitrite
concentration

On the other hand, the pH in polarographic cell has to be about

Fig. 1. Decomposition of nitrite with time. a) 10 mL 4 M HCl (10 min nitrogen). b)
a 0.1 mL 2
102 M NO 2 . c) After 4 min d) After 8 min.
According to the equation 1 mol of As (III) will react with 2 mol
of nitrite. The arsenite peak will decrease after the reaction with
nitrite and from this decrease the quantity of nitrite will be deter-
mined by standard addition of arsenite. This redox reaction has
been used rst time in this work.
3.3. Electrochemical behavior of As (III)
In various electrolytes the peaks of As (III) have been studied.
The results obtained are summarized in Table 1. As can be seen the
highest peaks were obtained at about pH 1.5. Three peaks were
observed in this medium. The rst peak at about 0.52 V is sharp, it
belongs to the reduction of As (III) into As, the peak at
about 0.67 V is also sharp but it does not show proportional in-
crements to standard additions since it is an adsorption peak
(experimentally proven) [21], and the third broad one at
1.5, since the highest peak of As (III) was obtained at about this pH
1.5. For the determination of arsenic the rst peak of As (III) has to
be used.
It is also important that there should be no peak overlap be-
tween nitrite peak and As (III) peak. For this purpose nitrite was
added into a solution (pH 1.5) containing As (III). As can be seen
from Fig. 2 the nitrite peak at about e 0.90 V and the third peak of
As (III) at 0.85 V overlapped. It can be concluded that the rst peak
of As (III) is suitable for the calculation. As expected there is no
decrease in As (III) peak by the addition of nitrite into polarographic
cell (pH 1.5) since the pH is not high enough so that the redox
reaction can take place.
3.5. Synthetic sample
A sample containing 1.0
103 M NO 2 4.0
10
3
M As (III) is
prepared at pH 5e7 in a reaction vessel, nitrogen gas was passed for
about 5 min so that the reaction has ended. A small sample of
0.1 mL was added (cell concentration 1
105 M) 2 times into a
polarographic cell containing 1 M HAc buffer (pH 1.5). The
polarograms, Fig. 3, are taken and then after standard additions of
As(III), the calculations are made from the peak heights at 0.52 V.

Table 1
Polarographic behavior of As(III) in various electrolytes.

Electrolyte Peak potential E (V) Peak height (mm) Peak shape

1 M AcOH/AcO (pH 1.4) 0.52/0.69/0.85 115/135/41 sharp/sharp/broad

1 M AcOH/AcO (pH 2.0) 0.72/0.86 38/27 sharp/broad
0.1 M HClO4 0.60/0.68/0.77 32/43/32 broad/sharp/broad
0.1 M H3PO4 0.62/0.72/0.86 31/44/26 broad/sharp/broad
0.1 M HCl 0.61/0.68/0.82 24/37/24 broad/sharp/broad
82 G. Somer et al. / Nitric Oxide 57 (2016) 79e84
Fig. 2. Interference of As(III) peaks with nitrite peaks. a) 10 mL (pH 1.4) 1 M HAc/
Ac. b) a 0.3 mL 1
103 M As(III). c) b 0.4 mL 1
103 M As(III). d) c 0.2 mL
1
103 M NO 2 . e) d 0.2 mL 1
10
3
M NO 2 . f) e 0.4 mL 1
10
3
M NO
2.
The result found was (0.98 0.2) x 105 M for N 4 and 90%
condence interval in the same medium. The limit of detection LOD
was calculated as 2
107 M for (S/N 3) for nitrite.
3.6. Interference studies
The interference of most common ions such as Fe (III), Pb (II), Zn
(II), Cu (II) and Cd (II), and anions such as nitrate, chloride and
sulfate ions were investigated. This kind of comprehensive inter-
ference work is not found in any former studies. Two aspects of the
interference of cations were studied. The rst is because of over-
lapping peaks with As (III) and the second is because of their
reduction potentials to see the possibility of reaction with As (III)
and nitrite. These are discussed in two sections below.
3.7. Interference because of overlapping peaks
To see whether there might be any interference, the peak po-
tentials have been determined by taking polarograms at pH 1.4. The
peaks of Fe (III), Cu (II), Pb (II), Cd (II), and Zn (II) were
at 0.05 V, 0.18 V, 0.42 V, 0.60 V, and 0.95 V, respectively. As
(III), on the other hand has peaks at 0.52 V, 0.67 V and 0.85 V. It
was found that Pb peak at 0.42 V overlaps with the rst As(III)
peak at 0.52 V, only when its concentration was 1/10 th or larger
than As(III) concentrations Fig. 4. Here we have to mention that,
that much lead cannot be present in food stuff since it is a poison. In
case of higher concentration of Pb in a sample, its peak potential
can be shifted by the addition of EDTA, since As (III) does not form a
complex its peak potential will not change [22].
Since sulfate, chloride, and nitrate ions are not electroactive,
they will not have a peak overlap.
Fig. 3. Determination of 1
105 M nitrite using As(III). a) 10 mL (pH 1.4) 1 M HAc/
Ac. b) a 0.1 mL sample. c) b 0.1 mL sample. d) c 0.1 mL 4
103 M As(III). e)
d 0.1 mL 4
103 M As(III). Sample: 1
103 M NO2 and 4
10
3
M As(III), pH 6.5
(5 min N2).
3.8. Interference because of standard reduction potentials
These potentials are important because of the possible redox
reaction with As (III). The standard reduction potentials are 0.77 V
for Fe (III), 0.34 V for Cu (II), 0.12 V for Pb (II), 0.76 V for Zn (II),
and 0.58 V for As (V). According to these data, only a reaction
between Fe (III) and As (III) may take place under suitable condi-
tions. Its effect have been studied at pH 2.5 and pH 6 by adding
Fe (III) into As (III) solution. No decrease in As (III) peak was
observed even when 25 times higher concentration of Fe (III) was
added. There will be no reaction between Fe (III) and nitrite because
of their unsuitable reduction potentials, while the reduction po-
tential of nitrite is 10 V and it is 0.77 V for Fe (III).
3.9. Application to sausage sample
A sausage sample of about 42 g was taken, it was whipped in
blender with 100 mL distilled water. It is ltered through cotton
gauze. After measuring its volume, the pH was adjusted to 5e7, and
a known quantity of As(III) was added, then waited for about 5 min
in order the reaction has ended, a sample of 0.1 mL was taken and
added into the polarographic cell containing 10 mL pH 1.5 acetate
buffer. The polarogram is taken, the quantity of nitrite was deter-
mined as KNO2 from the As(III) which was left over. Fig. 5. The
result found was 0.26 mg 0.02 mg (in 1 g sausage) for N 3 and
90% condence interval. The result obtained was quite similar
which was obtained by using sulte for the reaction [20].
 G. Somer et al. / Nitric Oxide 57 (2016) 79e84
Fig. 4. Overlap of Pb peak with As (III) peaks. a) 10 mL (pH 1.4) 1 M HAc/Ac. b)
a 0.1 mL 4
103 M As(III). c) b 0.3 mL 1
104 M Pb(II). d) c 0.4 mL
1
104 M Pb(II).
Fig. 5. Determination of nitrite in sausage. a) 10 mL (pH 1.4) 1 M HAc/Ac. b)
a 0.1 mL 4
103 M As(III). c) b 0.1 mL sample. d) c 0.1 mL 4
103 M As(III).
83
4. Conclusions
Electrochemical methods are mostly preferred for trace analysis
and speciation analysis because of their high selectivity and very
low detection limits without necessitating tedious extraction or
preconcentration procedures. The results obtained with DPP are
very reproducible, since with the use of a dropping mercury elec-
trode, the behavior of the electrode is independent of its past
history.
Nitrite salts of sodium or potassium are being used for the
protection of meat products. While they provide color and taste of
meat they may form carcinogenic substances. Thus, nitrite con-
centration in food-stuff has to be controlled carefully by highly
sensitive methods.
For the determination of trace nitrite, an indirect differential
pulse polarographic method has been developed. For this purpose,
As (III) has been used for the reduction of nitrite. The redox reaction
had to be made at pH values about 5e7 since at lower pH values
nitrite was decomposing. In order to have the reaction quantitative
As (III) concentration had to be at least 4 times or larger of nitrite
concentration, the reaction medium had to be stirred for about
5e10 min with nitrogen gas in order to expel the NO gas formed.
For the determination of nitrite, the As (III) quantity had to be
determined from its peak at about 0.52 V. The most suitable
electrolyte in polarographic cell was found as HAc, pH 1.5, so that
the As(III) peak will be sharp and high enough. Using this method it
was possible to determine low concentrations of nitrite. The LOD
found for nitrite was 2
107 M for (S/N 3). No interference was
observed from most common cations and anions.
This proposed method can be applied safely for many biological
samples which contain nitrite. In the presence of ascorbic acid also,
there will be no reaction with As(III) because of their standard
reduction potentials and pH used [23]. For this purpose the nitrite
content of sausage was determined with the proposed method. The
result found was very similar where sulte was used [20] for the
reduction of nitrite.
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