Spectrofluorimetric determination of trace nitrite with a novel fluorescent probe

Article abstract of DOI:10.1016/j.saa.2009.03.018

A simple, sensitive and selective fluorimetric determination for trace nitrites was developed using an unsymmetrical rhodamine with a high fluorescence quantum yield and large Stokes shift. The method is based on the reaction of the unsymmetrical rhodamine with nitrite in an acidic medium to form a nitroso product, which has much lower fluorescence because the electron-withdrawing effect of the nitroso group influences the system of p-π conjugation between N atom and benzene ring. Under optimum conditions, the fluorescence quenching intensity is linear over a nitrite concentration range of 1.0 × 10^-8 to 3.5 × 10^-7 M. The detection limit is 2.0 × 10^-10 M (S/N = 3). The method was applied to the determination of nitrite in tap water and lake water with satisfactory results. The mechanism for the reaction is also reported.

Full text of DOI:10.1016/j.saa.2009.03.018

Spectrochimica Acta Part A 73 (2009) 789–793
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Spectrofluorimetric determination of trace nitrite with a novel fluorescent probe
Qing-Hao Liua, Xi-Long Yana, Jin-Chun Guob, Dong-Hua Wangc, Lei Lia,
Fan-Yong Yand, Li-Gong Chena,∗
a School of Chemical Engineering and Technology, Tianjin University, Weijin Road, Tianjin 300072, China
b Qinghai Institute of salt lakes, Chinese Academy of Sciences, Xining 810008, China
c School of Pharmaceuticals and Biotechnology, Tianjin University, Tianjin 300072, China
d School of Material Science and Chemical Engineering, Tianjin Polytechnic University, Tianjin 300160, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A simple, sensitive and selective fluorimetric determination for trace nitrites was developed using an
unsymmetrical rhodamine with a high fluorescence quantum yield and large Stokes shift. The method is
based on the reaction of the unsymmetrical rhodamine with nitrite in an acidic medium to form a nitroso
product, which has much lower fluorescence because the electron-withdrawing effect of the nitroso
group influences the system of p-␲ conjugation between N atom and benzene ring. Under optimum
conditions, the fluorescence quenching intensity is linear over a nitrite concentration range of 1.0 × 10−8
to 3.5 × 10−7 M. The detection limit is 2.0 × 10−10 M (S/N = 3). The method was applied to the determination
of nitrite in tap water and lake water with satisfactory results. The mechanism for the reaction is also
reported.
Received 22 October 2008
Received in revised form 8 March 2009
Accepted 18 March 2009
Keywords:
Spectrofluorimetric
Nitrite
Fluorescent probe
Unsymmetrical rhodamine
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
methods, nitrite ions are either chemically reactive or serve as
a catalyst for a chemical reaction, the reaction products then
Nitrite is not only a very important nitrosating agent used
to form nitroso compounds, but also an excellent indicator
of the extent of pollution in environmental samples. Further-
more, nitrite salts, which are used as food preservatives, exist
widely in the environment, and they can cause various types
of noxious effect, such as microsomal enzyme inhibition, and
a decrease in the efficiency of a nutritive diet [1,2]. Therefore,
the determination of nitrite in water, food and biological and
environmental matrices is of vital significance, and in fact a sen-
sitive and accurate method for the determination of nitrite ions is
urgent.
Many analytical methods for nitrite have been reported
previously, including electrometry [3–5], chromatography [6,7],
capillary electrophoresis [8,9], spectrophotometry [10–12] and
spectrofluorimetry [13–20]. However, not all are suitable for rou-
tine ultra-trace determinations. Spectrophotometric methods [21]
suffer from poor sensitivity and interferences from other anions.
Chromatographic methods [22] are often time-consuming. Cap-
illary electrophoresis [23] is complicated and uses high-cost
instrumentation. Spectrofluorimetry is the most widely employed
method for detecting nitrites due to its simplicity, sensitivity,
excellent limits of detection and low-cost. In spectrofluorimetric
have an effect on the fluorescence properties (development,
inhibition, enhancement) in either a direct or indirect man-
ner [24]. Rhodamine has been widely used in biochemistry and
molecular biology because of its good stability, high extinc-
tion coefficient and large fluorescence quantum yield [25]. In
this paper, two unsymmetrical rhodamines with high fluores-
cence quantum yields and large Stokes shifts were synthesized
(Fig. 1). They were then tested as fluorescent probes for the
determination of nitrite. 6-(2-Carboxyphenyl)-9-(dimethylamino)-
3,4-dihydro-2H-chromeno[3,2-g]quinolin-1-ium (1) was chosen
to detect trace nitrite because of higher fluorescence quantum
yield.
2. Experimental
2.1. Apparatus
A RF-5000 spectrofluorimeter (Shimadzu) and Cary Eclipse
spectrofluorimeter (Varian) equipped with
quartz cell were used for the fluorescence measurements.
UV–vis absorption spectra were measured on computer-
controlled Shimadzu UV-2450 spectrometer. 1H NMR was
performed on INOVA 500, BRUKER AV400 and BRUKER
a
1 cm × 1 cm
a
a
∗
AV300 spectrometer using tetramethylsilane as the internal
reference.
Corresponding author at: Tel.: +86 22 27406314; fax: +86 22 27406314.
E-mail address: lgchen@tju.edu.cn (L.-G. Chen).
1386-1425/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2009.03.018

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Q.-H. Liu et al. / Spectrochimica Acta Part A 73 (2009) 789–793
(d, J = 9.3 Hz, 1H), 7.26 (d, J = 6.9 Hz, 1H), 7.69–7.76 (m, 2H), 8.21
(d, J = 6.6 Hz, 1H).
2.3.4. 2-Carboxyl-4ꢀ-diethylamino-2ꢀ-hydroxy benzophenone (5)
and 6-(2-carboxyphenyl)-9-(diethylamino)-3,4-dihydro-2H-
chromeno[3,2-g]quinolin-1-ium (2)
Compound 5 was prepared similarly to compound 4 in 73.1%
yield.
1H NMR (500 MHz, CD3OD:CDCl3 = 5:1): ı 1.15 (t, J = 7.0 Hz, 6H),
3.40 (q, J = 7.0 Hz, 4H), 6.09 (d, J = 2.5 Hz, 1H), 6.15 (d, J = 9.0 Hz, 1H),
6.84 (d, J = 9.5 Hz, 1H), 7.35 (dd, J = 7.5, 1.0 Hz, 1H), 7.58 (t, J = 7.5 Hz,
1H), 7.66 (t, J = 7.5 Hz, 1H), 8.03 (d, J = 8.0 Hz, 1H).
Compound 2 was synthesized using the same method as com-
pound 1 with a 74.6% yield.
Fig. 1. Molecular structures of compounds 1 and 2.
2.2. Reagents
All of chemicals were of analytical grade and used without fur-
ther purification. All solutions were prepared with doubly distilled
water.
1H NMR (300 MHz, CD3OD): ı 1.26 (t, J = 7.2 Hz, 6H), 1.85–1.96
(m, 2H), 2.66–2.67 (m, 2H), 3.23–3.32 (m, 2H), 3.60 (t, J = 7.4 Hz,
4H), 6.65 (s, 1H), 6.82–6.88 (m, 2H), 6.92 (d, J = 9.5 Hz, 1H), 7.08
(d, J = 9.3 Hz, 1H), 7.30 (d, J = 6.9 Hz, 1H), 7.69–7.77 (m, 2H), 8.24 (d,
J = 6.6 Hz, 1H).
A 2 × 10−5-M stock solution of compound 1 was prepared by
dissolving it in water. A standard nitrite solution (1 × 10−3 M) was
prepared by dissolving sodium nitrite in water, which was dried
at 100 ◦C for 2 h. Twenty drops of chloroform (about 0.3 mL) and a
pellet of sodium hydroxide (about 5.0 mg) were then added [26].
This standard solution was prepared weekly and kept in a refriger-
ator. Each day a new working solution was prepared by appropriate
dilution of the standard solution. Hydrochloric acid (0.1 M) was
prepared from concentrated hydrochloric acid.
2.4. Determination of nitrite
A nitrite test solution was prepared by adding 1.0 mL of
2 × 10−5 M compound 1 solution and 2.0 mL of 0.1 M HCl to a 10.0-
mL volumetric flask. A certain volume of the standard solution
of nitrite was then added and the resulting solution was diluted
to 5.0 mL with water. Then the solution was allowed to stand for
30 min at 30 ◦C, and finally diluted to 10.0 mL with water. The flu-
orescence intensity was measured at 561 nm with excitation at
538 nm. The slit width was 5 nm for both excitation and emission.
2.3. Synthesis of new fluorescence probes
The synthetic route for the new probes is shown in Fig. 2.
2.3.1. 7-Hydroxy-1,2,3,4-tetrahydroquinoline (3)
3. Results and discussion
Compound 3 was prepared according to the method of Kulka
and Manske [27] in the yield of 25.4%.
1H NMR (500 MHz, CDCl3): ı 1.85 (q, J = 6.0 Hz, 2H), 2.61 (t,
J = 6.0 Hz, 2H), 3.15–3.18 (m, 2H), 4.65 (br, 2H), 5.96 (d, J = 3.0 Hz,
1H), 6.03 (dd, J = 9.0, 3.0 Hz, 1H), 6.64–6.65 (m, 1H).
3.1. Spectral properties of new fluorescence probes
The maximum excitation wavelength of compound 1 is at
538 nm (ε = 5.40 × 104 cm−1 M−1) and its emission wavelength was
at 561 nm. While the maximum excitation wavelength of com-
pound 2 is at 542 nm (ε = 5.40 × 104 cm−1 M−1) with the emission
wavelength at 561 nm. Stokes shifts of compounds 1 and 2 are
762.0 and 708.2 nm, respectively. Fluorescent quantum yields of
compounds 1 (˚ = 0.79) and 2 (˚ = 0.73) were measured by using
rhodamine B (˚ = 0.5 in ethanol) [28] as the standard. Compound
1 has a higher fluorescent quantum yield and larger Stokes shift
than compound 2. The reason is that the diethylamino group of
compound 2 has more torsional motion which causes more loss
of energy than compound 1. Therefore, compound 1 was used to
detect nitrites.
2.3.2. 2-Carboxyl-4ꢀ-dimethylamino-2ꢀ-hydroxy
benzophenone (4)
A solution of 3-dimethylamino phenol (4.11 g, 30.0 mmol) and
phthalic anhydride (4.66 g, 31.5 mmol) in toluene (30 mL) was
refluxed under N2 for 3 h, and cooled to 50–60 ◦C. Then 30 mL of
35% aqueous NaOH (w/w) was added and heated at 90 ◦C for 6 h.
The resulting mixture was poured into H2O (300 mL), acidified with
HCl (10.0 M), and allowed to stand at room temperature for 2 h. The
suspension was then filtered. The solid was recrystallized from a
mixture of water and methanol, and dried to afford the desired
product (5.93 g, 71.3%).
1H NMR (500 MHz, CD3OD:CDCl3 = 5:1): ı 3.04 (s, 6H), 6.13 (s,
1H), 6.18 (d, J = 7.5 Hz, 1H), 6.88 (d, J = 8.5 Hz, 1H), 7.36 (d, J = 7.5 Hz,
1H), 7.59 (t, J = 7.5 Hz, 1H), 7.68 (t, J = 7.5 Hz, 1H), 8.07 (d, J = 7.5 Hz,
1H).
The excitation and emission spectra of compound 1 are shown
in Fig. 3. The fluorescence intensity of compound 1 decreased dra-
matically when nitrite was added.
3.2. Effect of HCl concentration
2.3.3. 6-(2-Carboxyphenyl)-9-(dimethylamino)-3,4-dihydro-2H-
chromeno[3,2-g]quinolin-1-ium (1)
Since the reaction proceeds in acidic media, the proper HCl con-
centration must be selected to ensure compound 1 reacts with the
nitrites as completely as possible. Fig. 4 indicates the effect of HCl
concentration on the fluorescence quenching intensity (ꢀF), where
ꢀF is the fluorescence intensity difference between the absence and
the presence of nitrite. The optimal HCl concentration is 0.05 M.
A solution of compound 4 (0.28 g, 1.0 mmol) and compound
3 (0.15 g, 1.0 mmol) in 5 mL methanesulfonic acid was heated
under N2 at 85 ◦C for 14 h. The cooled mixture was then poured
into 20 mL of ice water, neutralized with saturated aqueous
Na2CO3, and finally filtered. The crude product was dried and puri-
fied by column chromatography on silica to afford compound 1
(0.29 g) with a yield of 71.8% (methanol:dichloromethane = 1:20,
Rf = 0.1).
3.3. Effect of compound 1 concentration
1H NMR (300 MHz, CD3OD): ı 1.86–1.96 (m, 2H), 2.67–2.68
(m, 2H), 3.15–3.41 (m, 8H), 6.63 (s, 1H), 6.83–6.93 (m, 3H), 7.12
The effect of the concentration of compound 1 on the fluores-
cent quenching by the formation of the nitroso derivatives was

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791
Fig. 2. Synthetic scheme for fluorescent probes 1 and 2.
studied and the results are shown in Fig. 5. The maximum fluo-
rescent quenching was obtained at 2 × 10−6 M of compound 1. This
concentration was then selected for further studies.
is 30 ◦C. The optimal reaction time is 30 min. The products were
stable for at least 5 h after their formation as judged by UV–vis
absorption spectroscopy.
3.4. Effects of reaction time and temperature
3.5. Linearity, sensitivity and precision
The effects of reaction time and temperature on the reaction
of compound 1 with nitrite are shown in Fig. 6. Longer times are
needed to reach maximum fluorescence quenching at low temper-
atures (20 ◦C) than at high temperatures (30–40 ◦C). The optimal
reaction temperature to achieve maximum fluorescent quenching
Under the optimized conditions, fluorescence spectra of com-
pound 1 in the presence of nitrites were shown in Fig. 7 in the
concentration range of 1.0 × 10−8 to 3.5 × 10−7 M. The fluorescence
quenching intensity was linear over nitrite concentration. Based on
it, a linear calibration curve can be constructed in this concentration
range. The concentration of nitrite can be calculated from the linear
Fig. 3. Fluorescence spectra of compound 1 and compound 1 in the presence
of nitrite. Ccompound 1 = 2 × 10−6 M; CHCl = 0.05 M; nitrite: 1 × 10−7 M; reaction time,
30 min; reaction temperature, 30 ◦C. 1, 2, excitation and emission spectra of com-
pound 1, respectively; 1ꢀ, 2ꢀ, excitation and emission spectra of compound 1 in the
presence of nitrite, respectively.
Fig. 4. Effect of HCl concentration. Ccompound 1 = 2 × 10−6 M; nitrite: 1 × 10−7 M; reac-
tion time, 30 min; reaction temperature, 30 ◦C.

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Q.-H. Liu et al. / Spectrochimica Acta Part A 73 (2009) 789–793
Table 1
Tolerance limits of different ions in the determination of 1.0 × 10−7 M nitrite.
Foreign ion
Tolerance limit (molar ratio)
100,000
Mg2+, Hg2+, Ca2+, K+, Na+, Mn2+, Zn2+, NH4+
,
,
Ba2+, NO3−, CH3COO−, SO42−, F−, CO3
−
C2O42−, Cl−, Ce3+, Al3+, Fe3+, Cu2+, Br−, Fe2+
50,000
5,000
3−
PO4
Tartrate, citric
Table 2
Analytical results of nitrite in samples with compound 1.
Sample
Nitrite added (␮M)
Found (␮M)
R.S.D. (%)
Recovery (%)
97.3
Tap water
0.00
0.10
0.0786
0.1551
2.06
1.82
Lake water
0.00
0.10
0.0850
0.1725
2.18
1.36
102.9
Fig. 5. Effect of compound 1 concentration. CHCl = 0.05 M; nitrite: 1 × 10−7 M; reac-
tion time, 30 min; reaction temperature, 30 ◦C.
regression equation: ꢀF = 2396.8c + 11.08 (r = 0.9995), where ꢀF is
the fluorescence quenching intensity and c is the concentration of
nitrite (␮M). The limit of detection (LOD) is evaluated using 3ꢁ/s,
where ꢁ is the standard deviation of the blank signals and s is the
slope of the linear calibration plot. The LOD for the determination of
nitrite was thus calculated to be 2.0 × 10−10 M. The relative standard
deviation (n = 10) is 0.69% and 0.53% at 5.0 × 10−8 and 1.0 × 10−7 M
nitrite, respectively. The repeatability of the method was confirmed
by analysis of a standard nitrite solution over a period of 7 days
(n = 5). The relative standard deviation was found to be 2.36% at
1.0 × 10−7 M.
In comparison with previously reported spectrofluorimetric
methods in detecting trace nitrite, such as 5-aminofluorescein [29],
4-hydroxycoumarin [17], acetaminophen [30], safranine [31], rho-
damine 110 [15], TMDABODIPY [32] and TMDCDABODIPY [13], the
current method is more sensitive and simple.
3.6. Effects of foreign ions
The effects of foreign ions (cations and anions) on the determi-
nation of 1.0 × 10−7 M nitrite were also studied, and the results are
summarized in Table 1. The tolerance limit is defined as the con-
centration of foreign ion causing at least a 4% relative error in the
fluorescence of the sample. Most of ions examined do not interfere
with the determination of nitrite.
Fig. 6. Effect of time and temperature. Ccompound 1 = 2 × 10−6 M; CHCl = 0.05 M; nitrite:
1 × 10−7 M. 20 ◦C (᭹), 30 ◦C (ꢀ), 40 ◦C (ꢁ).
Fig. 7. The fluorescence spectra of the system of compound
1 and nitrite.
Fig. 8. Absorbance spectra of compound 1 and compound 1 in the presence of nitrite.
Ccompound 1 = 2 × 10−6 M; nitrite: 2 × 10−8 M; reaction time, 30 min; reaction tem-
perature, 30 ◦C. 1, absorbance spectrum of compound 1; 2, absorbance spectrum of
compound 1 in the presence of nitrite.
Ccompound 1 = 2 × 10−6 M; CHCl = 0.05 M; nitrite (×10−6 M); reaction time, 30 min; reac-
tion temperature, 30 ◦C: (a) 0; (b) 0.01; (c) 0.025; (d) 0.04; (e) 0.07; (f) 0.095; (g) 0.12;
(h) 0.14; (i) 0.18; (j) 0.20; (k) 0.22; (l) 0.25; (m) 0.275; (n) 0.3; (o) 0.325; (p) 0.35.

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793
Fig. 9. Reaction of compound 1 with nitrite.
3.7. Sample analysis
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4. Conclusions
A new fluorescence probe, unsymmetrical rhodamine, was syn-
thesized and used in the spectrofluorimetric determination of trace
nitrite in tap water and lake water with satisfactory results. The
current method has the advantages of simplicity, good reproducibil-
ity, high sensitivity and selectivity for the determination of trace
nitrite. The mechanism for the fluorescence quenching involves the
formation of an electron-withdrawing nitroso group.
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