2-Amino-5,7-dimethyl-1,8-naphthyridine as a fluorescent reagent for the determination of nitrite

Article abstract of DOI:10.1016/j.saa.2006.03.036

A new fluorescent reagent 2-amino-5,7-dimethyl-1,8-naphthyridine (ADMND) was proposed for the determination of trace nitrite. The reaction is based on the diazotization of naphthyridine amine with nitrite to form a diazonium salt that hydrolyzed when boiling to give hydroxyl group substituted naphthyridine. Fluorescence quenching degree of ADMND by nitrite ion is linear in the nitrite concentration range of 1 × 10^-7 to 2.5 × 10^-6 mol l^-1 with a detection limit of 4.06 × 10^-8 mol l^-1. Reaction and determination acidity for nitrite is the same which made the method much simpler compared with the widely accepted fluorescence method with DAN as a fluorescence reagent.

Full text of DOI:10.1016/j.saa.2006.03.036

Spectrochimica Acta Part A 66 (2007) 586–589
2-Amino-5,7-dimethyl-1,8-naphthyridine as a fluorescent
reagent for the determination of nitrite
Tian Chena, Aijun Tonga,∗, Yanmei Zhoub
a Department of Chemistry, Tsinghua University, Beijing 100084, China
b Department of Chemistry, Henan University, Kaifeng, Henan 475001, China
Received 16 November 2005; received in revised form 27 March 2006; accepted 30 March 2006
Abstract
A new fluorescent reagent 2-amino-5,7-dimethyl-1,8-naphthyridine (ADMND) was proposed for the determination of trace nitrite. The reaction
is based on the diazotization of naphthyridine amine with nitrite to form a diazonium salt that hydrolyzed when boiling to give hydroxyl group
substituted naphthyridine. Fluorescence quenching degree of ADMND by nitrite ion is linear in the nitrite concentration range of 1 × 10−7 to
2.5 × 10−6 mol l−1 with a detection limit of 4.06 × 10−8 mol l−1. Reaction and determination acidity for nitrite is the same which made the method
much simpler compared with the widely accepted fluorescence method with DAN as a fluorescence reagent.
© 2006 Elsevier B.V. All rights reserved.
Keywords: 2-Amino-5,7-dimethyl-1,8-naphthyridine; Nitrite; Fluorescence quenching; Determination
1. Introduction
[6]. Among them, DAN is the most widely used reagent for
fluorescence determination of nitrite. But the reaction and deter-
mination processes are complicated, including requirement of
different pH conditions in the reaction and detection, poor water
solubility of the product, and time-consuming.
Nitrite is ubiquitous within environmental, food, indus-
trial and physiological systems. Highly carcinogenic N-
nitrosoamines could be formed by the reaction of nitrite with
secondary amines and amides, which are generally present in
meat and fish products, where nitrite is used as a food preserva-
tive. Nitrogen dioxide, which is one of the most important atmo-
spheric pollutants, can be dissolved in triethanolamine (TEA)
solution, and exists as nitrite [1]. It is recommended that the
nitrite level in drinking water should not exceed 0.2 mg l−1 [2].
Thus, developing sensitive, specific, simple and low-cost meth-
ods for the determination of nitrite is of great importance.
Various methods have been reported for the determination of
nitrite, including spectroscopic and electrochemical detection,
chromatography and capillary electrophoresis [3]. Among these
methods, spectrofluorimetry is widely used due to its high sen-
sitivity and selectivity.
2-Amino-5,7-dimethyl-1,8-naphthyridine (ADMND) is a
new reagent synthesized in our laboratory. It can be synthe-
sized by only one simple step and serve as a ratiometric flu-
orescent pH probe near neutrality [7]. This high-fluorescent
reagent can react with nitrite to form a low-fluorescent com-
pound, 2-hydroxyl-5,7-dimethyl-1,8-naphthyridine (HDMND),
which can also serve as a fluorescent pH probe. Such fluores-
cence quenching process leads us to set up a new method for
nitrite determination. In this paper, ADMND was proposed as a
new fluorescent reagent for nitrite determination. Reaction and
determination conditions were studied, the method has proven
to be much simpler compared with those using DAN-related
fluorescent reagents and has been successfully applied to the
determination of nitrite in tap water samples.
Fluorescence determination of nitrite is usually based
on the reaction with a fluorescent reagent, such as 2,3-
diaminonaphthalene (DAN) [4], 2,6-diaminopyridine [5],
4,4ꢀ,4ꢀꢀ,4ꢀꢀꢀ-tetrasubstituted amino aluminium phthalocyanine
2. Experimental
2.1. Apparatus
∗
All fluorescence measurements were made with a JASCO
FP-6500 spectrofluorimeter equipped with an 1 cm quartz cell.
Corresponding author. Tel.: +86 1062787682; fax: +86 1062770327.
E-mail address: tongaj@mail.tsinghua.edu.cn (A. Tong).
1386-1425/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2006.03.036

T. Chen et al. / Spectrochimica Acta Part A 66 (2007) 586–589
587
Scheme 1. Reaction between ADMND and nitrite.
Excitation and emission slits were set at 5 nm. The absorption
spectrum was performed on a JASCO V-550 UV–vis spec-
trophotometer with bandpass as 1 nm.
2.2. Reagents
All reagents were of analytical reagent grade. All solutions
were prepared with doubly distilled water.
Stock solution of nitrite (1.5 × 10−3 mol l−1) was prepared
by dissolving sodium nitrite (Beijing Chemical Reagent Co.,
China, dried at 105 ◦C for 2 h before use) in water. This nitrite
solution was prepared every 2 weeks and kept in a refrigera-
tor. The working solution was prepared freshly by appropriate
dilution.
ADMND and HDMND were synthesized as described previ-
ously [7,8]. A 1.5 × 10–3 mol l−1 ADMND stock solution was
prepared by dissolving the dye in 1 mol l−1 sulfuric acid and
diluted with water as required.
Fig. 1. Absorption spectra of ADMND (1) and ADMND in the presence of
nitrite (2). [ADMND] = 1 × 10−5 mol l−1, [nitrite] = 5 × 10−5 mol l−1
.
2.3. General procedure
quenching and blue shift of both absorption and emission spec-
tra was observed. Similar spectra change was also found when
ADMND serves as a ratiometric fluorescence pH probe [7].
The 1 ml of 1 × 10–5 mol l−1 ADMND and 1 ml of
2.5 mol l−1 H2SO4 were transferred into a 5 ml volumetric flask,
final concentration of the sulfuric acid is 0.5 mol l−1 and thus
[H+] = 1 mol l−1. Then a standard solution of sodium nitrite was
added and the resulting solution was diluted to 5 ml with water.
Theflaskwasplacedintoaboilingwaterbathfor20 minandthen
cooled to room temperature. The relative fluorescence intensity
was measured at 403 nm when excited at 346 nm.
3.2. Effect of reaction acidity
The reaction between ADMND and nitrite is a diazonium
procedure, which should occur in acidic medium. In the present
3. Results and discussion
3.1. Spectral characteristics and proposed mechanism
Fluorescence determination of nitrite with ADMND is typi-
cally based on the reaction of naphthyridine amino group with
nitrite to form a diazonium salt that hydrolyzed when boiling to
give hydroxyl group substituted naphthyridine HDMND. The
proposed reaction mechanism is shown in Scheme 1 as reported
in the literature when aromatic amine was used [3,7].
The absorption and fluorescence emission spectra of
ADMND and its reaction product HDMND are shown in
Figs. 1 and 2. The excitation and emission maxima were found
to be at 346 and 403 nm, respectively. Decrease of fluorescence
intensity, blue shift of the fluorescence spectra were found when
nitrite was added. In the absorption spectra, blue shift of the
absorption maxima was also found in the presence of nitrite.
As the electron donating power of hydroxyl group in HDMND
was weaker than the primary amine in ADMND fluorescence
Fig. 2. Fluorescence excitation and emission spectra of ADMND (1) and
ADMND in the presence of nitrite (2). [ADMND] = 1 × 10−5 mol l−1
,
[nitrite] = 5 × 10−5 mol l−1
.

588
T. Chen et al. / Spectrochimica Acta Part A 66 (2007) 586–589
Fig. 5. Effect of reaction time and temperature. [ADMND] = 1 × 10−5 mol l−1
,
Fig. 3. Effect of H+ concentration on fluorescence intensity change of ADMND
after reaction. [ADMND] = 1 × 10−5 mol l−1, [nitrite] = 5 × 10−5 mol l−1. Boil-
ing water bath for 60 min reaction. I0 and I refer to the fluorescence intensities
of ADMND solution before and after the reaction with nitrite ion.
[H+] = 1 mol l−1, [nitrite] = 5 × 10−5 mol l−1. I0 and I refer to the fluorescence
intensities of ADMND solution before and after the reaction with nitrite ion:
(ꢀ) room temperature and (᭹) boiling water bath.
HDMND was the largest when detecting at the pH around 4–6
and thus the detection sensitivity would be the highest in this pH
range. However, as can be seen in Fig. 4, the fluorescence inten-
sity of ADMND itself changes abruptly in this pH range. So it is
hard to control pH at a fix value in this pH range to get a stable
fluorescence emission, causing poor reproducibility. From Fig. 4
it is seen that fluorescence quenching of ADMND by HDMND
was also large enough when detecting at more acidic condition.
Detecting at the pH just the same as reaction condition in the
present work has some advantages like, much simpler determi-
nation procedure compared with the most reported works that
needed adjusting pH from acidic in the reaction condition to
basic for fluorescence detection [9,10], eliminating most of the
influence caused by foreign ions. In this work, the detecting pH
condition was chosen as 1 mol l−1 H+ solution as the same pH
condition of reaction and satisfied sensitivity was obtained as
indicated in Section 3.4.
work, we choose H2SO4 to obtain an acidic medium. To study
the effect of acidity on the reaction, the concentration of H+ was
varied in the range of 0.1–1.2 mol l−1. As can be seen in Fig. 3, a
final concentration of 1 mol l−1 H+ was chosen for the reaction.
3.3. Choice of detection pH
It was found that the fluorescence of ADMND was sensitive
to pH, especially near neutrality [7]. To study the pH dependence
of the reaction product of nitrite with ADMND, HDMND was
also synthesized. Fig. 4 shows the pH dependence of the fluo-
rescence intensity for both ADMND and HDMND. It is found
that the fluorescence intensity difference between ADMND and
3.4. Effect of reaction time and temperature
As shown in Scheme 1, the last step of reaction is the hydrol-
ysis of diazonium salt that needs a high temperature condition.
The effect of reaction time and the reaction temperature were
examined and the results are shown in Fig. 5. It is found that
Table 1
Effect of foreign ions
Ions
Tolerance limit
(molar ratio)
Mg2+, Zn2+, Cu2+, Li+, Mn2+, Fe2+, NH4+, K+, tartrate,
1000
SO42−, Cl−, EDTA, CO32−, Br−, HCOO−, B4O7
,
2−
−
CH3COO−, H2PO4
Fe3+
10
100
−
Fig. 4. pH dependence of ADMND (1) and HDMND(2). Concentration of 1
NO3
and 2 was the same, 2 × 10−7 mol l−1
.

T. Chen et al. / Spectrochimica Acta Part A 66 (2007) 586–589
589
Table 2
Determination of nitrite in water samples
Sample
Nitrite added (nmol)
Nitrite founda (nmol)
R.S.D. (%)
Recovery (%)
Deionized water
0.00
2.00
3.00
5.00
0.328
2.408
3.680
5.408
7.3
0.5
1.4
0.8
–
104
111
102
Tap water in Tsinghua Universityb
0.00
20.00
40.00
60.00
80.00
13.90
31.80
53.94
69.05
92.76
2.48
2.25
8.28
6.29
2.89
–
89.5
100.1
91.92
98.58
a
Average of three determinations.
In the presence of 1 × 10−3 mol l−1 EDTA.
b
samples were then reacted with ADMND in 1 mol l−1 H2SO4
according to the reaction and detection procedure described in
Section 2. The results are shown in Table 2. The final concentra-
tion of nitrite in deionized water and tap water are 6.56 × 10−7
and 1.39 × 10−5 mol l−1, respectively. Nitrite concentration in
tap water is about three times higher than the drinking water
tolerance [2] therefore it is not suggested to drink the tap water
in China directly except for stated specially.
a boiling water bath was necessary to get obvious fluorescence
response upon nitrite reaction. In sample analysis lower nitrite
concentrationwasexpectedandwhentheconcentrationofnitrite
is chosen as 1 × 10–5 mol l−1 20 min reaction in boiling water
bathwasenough. Thisconditionwasusedinthefollowingexper-
iments.
3.5. Effect of diverse ions
The effect of diverse ions on the fluorescence determina-
tion of nitrite with ADMND was studied and the results are
summarized in Table 1. The tolerance limit was taken as the
concentration of a diverse ion causing less than 5% relative
error on the nitrite determination. As can be seen in the table,
most of the usual ions have no influence on the determination
of 1 × 10−5 mol l−1 nitrite. Influence of high concentration of
iron(III) can be eliminated by addition of a masking agent like
EDTA and effect of nitrate can also be avoided by proper reduc-
tion to nitrite as reported [11].
4. Conclusion
Based on the pH dependent fluorescence property of
2-amino-5,7-dimethyl-1,8-naphthyridine (ADMND) and 2-
hydroxyl-5,7-dimethyl-1,8-naphthyridine (HDMND), a new
fluorescence reagent ADMND for nitrite determination was pro-
posed. The reaction of nitrite with ADMND and fluorescence
determination of the product HDMND were all performed in
0.5 mol l−1 H2SO4 solution, which greatly simplified the pro-
cess but still with sufficient detection sensitivity compared with
the existing method by using DANas a fluorescence reagent. The
method is simple and effective, and is expected to be in prac-
tical use in chip based in situ determination of nitrite, nitrogen
dioxide and related compounds.
3.6. Linearity, sensitivity, and precision
A linear calibration curve was obtained in the nitrite con-
centration range of 1 × 10−7 to 2.5 × 10−6 mol l−1 with cor-
relation coefficient R as 0.996. The correlation equation is
I0/I = 1.7 × 105 [nitrite] + 0.99 where I0 and I are the fluores-
cence intensities in the absence and presence of nitrite, respec-
tively, at 403 nm. The relative standard deviation (n = 3) is 0.23%
at 1 × 10−7 mol l−1 nitrite. With a signal to noise ratio of 3,
the detection limit is calculated as 4.06 × 10−8 mol l−1 which is
comparable to the existed fluorescence method with aromatic
amine but needs more complicated determination procedure
[9,12].
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3.7. Determination of nitrite in water samples
Thepresentmethodwasappliedtothedeterminationofnitrite
in water samples. Deionized water was analyzed directly by
transferring 0.5 ml of the water into a 5 ml volumetric flask. Tap
water was analyzed in the presence of 1 × 10−3 mol l−1 EDTA
to avoid the influence of Fe3+ and 1 ml sample was taken. The