Talanta 72 (2007) 1675–1680
Flow-injection spectrofluorometric determination of trace amounts of
formaldehyde in water after derivatization with acetoacetanilide
Qiong Li, Mitsuko Oshima, Shoji Motomizu ∗
Department of Chemistry, Faculty of Science, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan
Available online 1 February 2007
Abstract
A novel fluorophotometric method for formaldehyde determination in environmental waters was developed: the method does not require any
enrichment procedures. A flow-injection analysis method for the spectrofluorometric determination of formaldehyde in waters, which is based on the
reaction of formaldehyde with acetoacetanilide and ammonia, is proposed. The proposed method shows a good linearity from 0.50 to 40 × 10−7 M,
and the limit of detection (LOD) of 3 × 10−9 M (0.09 ppb) is achievable. The sample throughput is 15 h−1 . One of the main advantages in the
proposed method is that the reaction can be carried out at room temperature without any heating system. The effect of various interferences
possibly present in the real water samples was investigated. Most cations and anions, as well as organic compounds, do not interfere with the
determination of formaldehyde in environmental water samples. The proposed method is very simple, rapid, less expensive, and highly sensitive,
and can be applied to the environmental water samples, such as rain, tap water and river water, at low concentration levels without any enrichment
procedure.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Spectrofluorometric; Water samples; Flow-injection; Formaldehyde determination; Acetoacetanilide
1. Introduction
The organic compound, formaldehyde, is a gasous substance
with a pungent smell, and it is the simplest aldehyde. Formaldehyde is unfavorable for our health because at low concentration
levels, formaldehyde can cause irritation of eyes, nose, throat,
and skin. Further, people with asthma may be more sensitive to
the effects of inhaled formaldehyde. Therefore, formaldehyde is
one of the analytically interesting substances in aquatic and air
environment as an environmental pollutant. Although formaldehyde is a gas at room temperature, it is readily soluble in water. In
an aqueous media, formaldehyde can polymerize, and formalin
actually contains only a little formaldehyde in the form of H2 CO
monomer. Oral administration of large amounts of formaldehyde can cause severe pain, vomiting, coma, and possible death.
Formaldehyde can enter drinking water as a result of human
activities, major sources being the discharge of industrial wastes
∗
Corresponding author. Tel.: +81 86 251 7846; fax: +81 86 251 7846.
E-mail address: motomizu@cc.okayama-u.ac.jp (S. Motomizu).
0039-9140/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.talanta.2007.01.054
and oxidative water treatment processes such as ozonation and
chlorination [1].
Due to the influence of HCHO to nature and human bodies, a number of analytical methods have been proposed. Trace
amounts of formaldehyde have been commonly determined by
spectrophotometric methods [2–8]. However, some of them are
not sensitive enough for the analysis of real water samples and
are sometimes subject to numerous interferences, which are serious problems. HPLC with 2,4-dinitrophenylhydrazine (DNPH)
as a derivatization agent [9–12] is one of the most frequently
used methods. Recently, Burini and Coli [13] reported a HPLC
system coupled with a diode array detector for formaldehyde
determination after derivatization with ethyl 3-oxobutanoate:
the limit of detection (LOD) was 0.024 g ml−1 . HPLC procedures, however, are time-consuming and are less adaptable to
water samples.
The fluorometric methods based on the Hantzsch reaction,
which involve the cyclization of amine, aldehyde and β-diketone
to form a dihydropyridine derivative, have often been used
for the detection of formaldehyde in aqueous solutions. Nash
[14] introduced a colorimetric method into analytical chemistry for HCHO, which was based on the Hantzsch reaction
of formaldehyde with acetylacetone (AA) or 2,4-pentanedione
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Q. Li et al. / Talanta 72 (2007) 1675–1680
in the presence of ammonia to form a yellow product of 3,5diactyl-1,4-dihydrolutidine (DDL). Later, Belman [15] found
that without any other changes, highly sensitive measurement
could be made by fluorometry, instead of spectrophotometry.
This detection reaction with AA gave less product of lutidine
with all aliphatic aldehydes except formaldehyde because of the
mildness of the reaction conditions of analysis. However, this
method is in general time-consuming and needs high temperature. Later, Sawicki and Carnes [16] proposed other reagents for
the fluorometric detection of formaldehyde: 5,5-dimethyl-1,3cyclohexnedione (dimedone) and 1,3-cyclohexanedione (CHD).
Both of them can offer excellent sensitivity for the detection of
HCHO, though such reactions require high temperature for the
reaction, and furthermore, the CHD methods can suffer from
positive interference from H2 O2 [17]. Aiming at developing
sensitive methods of analysis for HCHO, flow-injection analysis (FIA) has been frequently used. Fluorometric FI methods
have been studied with 4-amino-3-penten-2-one (Fluoral P) [18],
1,3-cyclohexanedione (CHD) [19], 5,5-dimethylcyclohexane1,3-dione (dimedone) [20], acetylacetone [21] and acridine
yellow-bromate [22]. In addition, Li et al. [23] developed an FI
chemiluminescence method with bromate–rhodamine 6G system, which showed a detection limit of 0.3 g l−1 (0.3 ppb).
However, they are simple but are not sensitive enough and are
subject to interferences from other compounds.
In Japan, the maximum concentration of HCHO, which is
allowed in drinking water, is now 80 ppb (2.7 M). In general, concentrations of HCHO in tap waters range from 0.15 to
15 ppb in Japan [1], and therefore, any enrichment procedures
are always necessary for the preconcentration of formaldehyde
before measurement [1,24,25]. Now, a simple and highly sensitive method for formaldehyde determination is required for the
direct analysis of water samples without any preconcentration
techniques.
Recently, the authors examined several detection reactions
for formaldehyde based on the Hantzsch reaction using novel
reagents, which were benzoylacetone, N-methylacetoacetamide,
n-acetoacetyl-o-toluidine and acetoacetanilide (AAA) [26]. Of
these, AAA was found to be most reactive, selective and sensitive
by a batchwise/spectrofluorometry. Furthermore, AAA is more
sensitive than the ones reported so far.
In this paper, a novel detection reagent, AAA, for the determination of formaldehyde is proposed for the flow-injection
method coupled with spectrofluorometry: the detection is based
on the Hantzsch reaction, which involves the cyclization
between AAA and formaldehyde in the presence of ammonium acetate. The reaction conditions were optimized by a
flow-injection method, and the method was applied to the determination of trace amounts of formaldehyde in environmental
waters.
Japan) was used throughout the experiments for the preparation of all solutions. A 0.2 M acetoacetanilide stock solution
was prepared by dissolving 3.544 g of acetoacetanilide (Wako
Pure chemicals, Osaka) in 50 ml of ethanol and diluting it to
100 ml with the purified water. An ammonium acetate stock
solution was prepared by dissolving 77.10 g of ammonium
acetate (Wako Pure chemicals, Osaka) in the purified water
and diluting it to 250 ml with the purified water. A 0.10 M
standard solution of formaldehyde was prepared by diluting
0.78 ml of 36.0–38.0% HCHO solution (Wako Pure chemicals,
Osaka) to 100 ml with purified water, followed by an accurate concentration determination using the iodometric method.
The working standard solutions were daily prepared by accurate dilution of the standard stock solution. For interference
testing, the following compounds were used: sodium chloride, sodium nitrate, sodium nitrite, sodium sulfate, sodium
sulfite, sodium carbonate, copper(II) chloride, iron(III) nitrate,
hydrogen peroxide, acetic acid, acetone, propionaldehyde and
acetaldehyde.
2.2. Apparatus
A schematic diagram of a flow-injection analysis (FIA) system is shown in Fig. 1. A double-plunger micropump (Sanuki
Kogyo, RX-703T, Japan), P, was used for propelling a carrier solution (CS) and a reagent solution (RS). A six-way
valve (Sanuki Kogyo, Japan), V, was used for introducing
standard formaldehyde solutions and samples into the carrier
stream. A 0.5 mm i.d. PTFE tubing was used for flow lines.
A thermostating dry bath (Iuchi, EB-303, Japan) was used
throughout the whole experiment. Peaks for HCHO determination were measured at λex = 370 nm and λem = 470 nm by
a fluorescence detector (Shimadzu, RF-10A XL, Japan) with
a micro flow-though cell (16 l). Peak height was recorded
with a chart strip recorder (TOA, FBR-251A, Japan). All measurements were performed in a temperature-controlled room
(25.0 ± 0.1 ◦ C).
2.3. Flow-injection procedure for the determination of
HCHO in aqueous solutions
The manifold of the flow-injection system used in this work
is shown in Fig. 1. In the proposed method, the flow rate of the
2. Experimental
2.1. Reagents
All chemicals used in this work were of analytical reagent
grade, and the water purified with a Milli Q Labo (Millipore,
Fig. 1. FIA system for the determination of HCHO using acetoacetanilide as a
reagent. CS: carrier solution (distilled water); RS: 0.05 M acetoacetanilide and
2.0 M ammonia acetate solution in 40% ethanol solution (pH 7.5); P: pump RX703T; V: six ways valves with 300 l loop; RC: reaction coil (10 m × 0.5 mm
i.d.); D: fluorometric detector FP-920; R: recorder; temperature: 25 ◦ C.
Q. Li et al. / Talanta 72 (2007) 1675–1680
1677
carrier and the reagent solution was set up at 0.3 ml min−1 . A
six-way valve with a sample loop (300 l) was used for introducing the working standard solutions of HCHO into the carrier
stream for the preparation of a calibration graph. The standard HCHO solution was mixed with the reagent solutions, and
flowed into the reaction coil (RC). Fluorescence changes of the
reaction product were measured with the fluorescence detector:
an excitation and an emission wavelength were 370 and 470 nm,
respectively. The flow signals were recorded with a chart strip
recorder.
3. Results and discussion
3.1. Optimization manifold parameters for
spectrofluorometric determination of formaldehyde
Fig. 3. Effect of flow rate. HCHO concentration () 0 (blank); () 1 × 10−6 M;
() 2 × 10−6 M; (䊉) 4 × 10−6 M.
The optimization of manifold parameters was performed
using the FIA manifold with fluorometric detector in Fig. 1. The
effect of reaction temperature on signal intensity was examined
by varying the temperature from 25 to 80 ◦ C using the dry heating bath. The results obtained are shown in Fig. 2. The results
showed that at temperature of 50 ◦ C gave the strongest intensity, while above 50 ◦ C, the baseline is not stable and some air
bubbles occurred. When higher sensitivity is required, heating
system can be used. However, for convenient operation, 25 ◦ C
(room temperature) was selected as a compromise between
the sensitivity and the convenience of the flow system in this
work.
At room temperature, reaction time is very important for
improving the reaction efficiency of the detection reaction. The
effect of the flow rate of the carrier and the reagent solution was investigated in the range of 0.2–0.6 ml min−1 . The
results obtained (Fig. 3) indicates that with increasing flow
rate from 0.2 to 0.6 ml min−1 , the sensitivity of the detection of HCHO is lowered though the sampling frequency is
higher. Considering the sensitivity and the sample throughput,
0.3 ml min−1 of the flow rate was chosen in the further experiments.
The effect of mixing coil length was examined by varying the
length from 4 to 14 m. As shown in Fig. 4, the signal intensity
increased with increasing the mixing coil length up to 10 m, and
Fig. 4. Effect of mixing coil length. HCHO concentration () 0 (blank); ()
1 × 10−6 M; () 2 × 10−6 M; (䊉) 4 × 10−6 M.
Fig. 2. Effect of reaction temperature. HCHO concentration () 0 (blank); ()
1 × 10−6 M; () 2 × 10−6 M; (䊉) 4 × 10−6 M.
Fig. 5. Effect of sample volume. HCHO concentration () 0 (blank); ()
1 × 10−6 M; () 2 × 10−6 M; (䊉) 4 × 10−6 M.
above 10 m, signal intensity was almost identical. A reaction
coil length of 10 m was chosen as a compromise with respect of
the sensitivity and the sample throughput.
The sample injection volumes of 100, 200, 300, 400 and
500 l were examined by changing the length of the sample
loop of the injection valve. From the results obtained (Fig. 5), it
can be seen that larger volumes were preferable to obtain higher
peak, and the volumes above 300 l gave only a small increase
in peak height. The sample volume of 300 l was selected as
a compromise of the sensitivity, the sample throughput and the
sample size.
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Q. Li et al. / Talanta 72 (2007) 1675–1680
Fig. 6. Effect of concentration of acetoacetanilide. HCHO concentration () 0
(blank); () 1 × 10−6 M; () 2 × 10−6 M; (䊉) 4 × 10−6 M.
Fig. 8. Effect of concentration of ammonium acetate. HCHO concentration ()
0 (blank); () 1 × 10−6 M; () 2 × 10−6 M; (䊉) 4 × 10−6 M.
3.2. Optimization of reagent concentrations for
spectrofluorometric determination of formaldehyde
Ammonium acetate works as both buffer and one of the
components of the reagents in the proposed method. The
effect of ammonium acetate concentration was examined in
the range of 0.5–2.5 M. The results obtained are shown in
Fig. 8. It was found that the peak height increased with
increasing ammonium acetate concentration. In the proposed
method, 2.0 M ammonium acetate concentration was selected
because of stronger capacity, higher sensitivity and better baseline.
In the reaction of formaldehyde with the proposed reagents,
pH of the reagent solution is very important for the reaction
efficiency. The effect of pH on the sensitivity was investigated
in the range of pH 5.0–8.0 using ammonium acetate as buffers:
the pH was adjusted with acetic acid and NaOH solution. The
results obtained are shown in Fig. 9, which indicates that in
the pH range over 6.5–7.5, the peak height is almost identical,
and below pH 6.5 and above pH 7.5, the peak height becomes
shorter. From these results, the pH of 7.5 was chosen for further
experiments.
In this work, acetoacetanilide was proposed for formaldehyde detection based on Hantzsch reaction. Two molecules of
an acetoacetanilide were initially involved in the unprecedented
transformation: one reacts with formaldehyde and the other one
reacts with ammonia to form an enamine-type intermediate,
followed by a cyclodehydration to afford the dihydropyridine
derivative [26]. Therefore, the effect of acetoacetanilide concentrations in the range of 0.02–0.08 M on the fluorescence intensity
was studied. The results in Fig. 6 indicates that the peak height
increased with increasing acetoacetanilide concentration up to
0.05 M, above which the signal intensity was almost identical.
In this study, 0.05 M acetoacetanilide was selected.
Experimental results demonstrated that the reaction could
proceed better in some organic solvents. A comparison of
ethanol, methanol and acetone was examined. The results
implies that higher and more constant sensitivity could be
obtained in ethanol solution medium. Therefore, the effect of
ethanol concentration in the range of 10–50% on fluorescence
intensity was studied. Fig. 7 shows that with increasing concentration of ethanol, the peak height increased. However, high
reagent blank and noisy background was obtained when ethanol
concentration was more than 40%. Therefore, 40% ethanol solution was chosen for the further experiments.
Fig. 7. Effect of concentration of ethanol. HCHO concentration () 0 (blank);
() 1 × 10−6 M; () 2 × 10−6 M; (䊉) 4 × 10−6 M.
3.3. Interference from foreign substances
The investigation of possible interferences was conducted
with regard to possible chemical interferences and the problem
of selectivity. The interference from low-molecular-weight aldehydes and other substances was investigated. It was found that
their interferences with the determination of HCHO in water
Fig. 9. Effect of pH. HCHO concentration () 0 (blank); () 1 × 10−6 M; ()
2 × 10−6 M; (䊉) 4 × 10−6 M.
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Q. Li et al. / Talanta 72 (2007) 1675–1680
Table 1
Tolerable concentrations of foreign species for the determination of 1 × 10−6 M
HCHO
Foreign substances
Na+
Cl−
H2 O2
NO3 −
HAc
NO2 −
SO4 2−
Acetone
Propionaldehyde
CO3 2−
Acetaldehyde
Cu2+
Fe3+
SO3 2−
Tolerable
concentration
(M)
Tolerable limit
([species]/[HCHO])
Relative
error (%)
1 × 10−2
1 × 10−2
1 × 10−2
1 × 10−2
1 × 10−2
5 × 10−3
2 × 10−3
2 × 10−3
1 × 10−3
1 × 10−3
5 × 10−4
2.5 × 10−5
1 × 10−5
2 × 10−6
10,000
10,000
10,000
10,000
10,000
5,000
2,000
2,000
1,000
1,000
500
25
10
2
+4.2
+4.2
−4.2
+4.7
−3.7
−4.4
−3.8
+3.7
+4.2
+4.7
+3.0
+2.7
+4.3
−3.8
samples were negligible even when interfering substances were
added at higher concentrations than commonly existing ones.
However, the HCHO signal decreased seriously with the addition of more than two-fold of sulfite ion. This interference is due
to the reaction of HCHO with sulfite. Since sulfite is easily oxidized to sulfate, only a low concentrations of sulfite can remain
in natural waters. Therefore, the proposed method is free from
interference with the determination of formaldehyde in environmental waters. In Table 1, the tolerable concentration is defined
as the concentrations of foreign substances causing less than
±5% relative error.
Table 2
Summary of the optimal conditions for the proposed method
Parameter
Range examined
Optimal
conditions
Reaction temperature (◦ C)
Flow rate (ml min−1 )
Mixing coil length (m)
Sample volume (l)
Acetoacetanilide concentration (M)
Ethanol concentration (%, v/v)
Ammonium acetate concentration (M)
pH
25–80
0.2–0.6
4–14
100–500
0.02–0.08
10–50
0.5–2.5
6.0–8.0
25
0.3
10
300
0.05
40
2.0
7.5
3.4. Calibration graph and analytical features
Under the optimal conditions summarized in Table 2, the calibration graph was prepared over the range of 0.50–40 × 10−7 M
HCHO with a correlation coefficient of 0.9999. The peak
profile of HCHO for the calibration graph obtained are
shown in Fig. 10. The equation of the calibration graph was
expressed as Y = 0.57X + 0.19, where Y is the peak height
and X is the HCHO concentration in 10−7 M. The standard
deviation and the relative standard deviation of 12 replicate
injections of 5.0 × 10−7 M were 0.052% and 1.7%, respectively.
The limit of detection, calculated as the concentration corresponding to three times of the baseline noise (S/N = 3), was
3 × 10−9 M (0.09 ppb). The proposed method is superior in
terms of sensitivity compared with other flow-injection spectrofluorometric methods as shown in Table 3.
Fig. 10. Flow signals for HCHO determination. HCHO concentration: 0–40 × 10−7 M; 0.05 M acetoacetanilide; 40% ethanol solution; 2.0 M ammonium acetate;
pH 7.5; flow rate: 0.3 ml min−1 ; reaction coil length: 10 m; sample injection volume: 300 l; reaction temperature: 25 ◦ C.
Table 3
Comparison of flow-injection spectrofluorometric method for the determination of formaldehyde
Reagent
4-Amino-3-penten-2-one (Fluoral P)
1,3-Cyclohexanedione (CHD)
5,5-Dimethycyclohexane-1,3-dione
(Dimedone)
Acetylacetone (AA)
Acetoacetanilide (AAA)
Reaction temperature (◦ C)
Working range (M)
Detection limit (M)
Sample throughput (h−1 )
Reference
60
95
130
0.5–100 × 10−6
–
6 × 10−9
3 × 10−8
12
–
20
[18]
[19]
[20]
8 × 10−9
3 × 10−9
20
15
[21]
This work
60
25
0.14–1.4 × 10−7
1.7–3.3 × 10−7 and
0.83–3.3 × 10−6
1.5–15 × 10−7
0.50–40 × 10−7
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Q. Li et al. / Talanta 72 (2007) 1675–1680
water and river water) at low concentration levels without any
enrichment procedure.
Table 4
Recovery tests of formaldehyde in environmental waters
Sample
Concentration
added (ppb)
Concentration
recovered (ppb)
Recovery
(%)
Acknowledgement
Rain
0
7.5
15.0
30.0
15.7
22.6
31.0
45.2
–
96.2
101.9
96.8
This work was supported partially by the Grant-in-Aid for
Scientific Research (B) (no. 16350044) from Japan Society for
the Promotion of Science (JSPS).
River water
0
1.5
3.0
6.0
3.53
5.25
6.83
9.67
–
106.2
108.4
104.0
Tap water 1
0
1.5
3.0
1.13
2.9
4.2
–
110.2
106.2
Tap water 2
0
1.5
3.0
1.29
2.91
4.36
–
107.6
105.4
All values are means (n = 4).
3.5. Application of the proposed method to environmental
water samples
The proposed method was applied to the determination of
formaldehyde in rain, river and tap water samples. All samples
were filtered using filter papers before introducing to the flowinjection system. In order to evaluate the validity of the proposed
method for the determination of formaldehyde, the recovery test
was performed. The samples were spiked with known amounts
of formaldehyde solutions from 1.5 to 30.0 ppb, which covers
the usual concentration ranges of formaldehyde in environmental water samples. Significantly high recoveries from 96.2% to
110.2% were obtained from the determination of formaldehyde
in water samples (Table 4).
4. Conclusions
A new reagent, acetoacetanilide, is first introduced for the
sensitive determination of trace amounts of formaldehyde.
The proposed method has several advantages over the previously reported methods: (1) simpler analysis system, (2) higher
sensitivity and (3) less toxicity of the reagents used.
The proposed method can be directly applied to the determination of HCHO in the environmental water samples (rain, tap
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