An intramolecular Mannich type reaction of ortho-amino aromatic azo dye and its detection effect for formaldehyde

Article abstract of DOI:10.1016/j.dyepig.2020.108376

A series of aromatic primary amine dyes were designed and applied for the modification of silk fibroin with Mannich-type reaction in the previous studies. Most aromatic primary amine dyes achieved the expected results with the participation of formaldehyde. However, those azo dyes with ortho-amino aromatic structure lost their colour and did not have any modification on silk fibroin under the same conditions of treatment. Therefore, the decolorization phenomenon of ortho-aniline aromatic azo dyes was analyzed using FT-IR, ESI-MS, NMR and Gaussian computation and the results indicated that an intramolecular Mannich reaction occurred between ortho-amino aromatic azo dyes and formaldehyde. In addition, a secondary effect of reduction of dyes by formaldehyde took place, in which the conjugated chromogenic system of the dye molecules was destroyed, producing benzimidazole, o-phenylenediamine and nitrogen-containing heterocyclic substances with polymethyl substitution. This decolorization of dye was studied as a detection or monitoring method for formaldehyde, and it showed advantages of sensitive response, clear visual indication, and no volatilization of organic solvent.

Full text of DOI:10.1016/j.dyepig.2020.108376

Journal Pre-proof
An intramolecular Mannich type reaction of ortho-amino aromatic azo dye and its
detection effect for formaldehyde
Pu Gao, Hua Jiang, Weiguo Chen, Zhihua Cui
PII:
S0143-7208(19)33022-0
DOI:
https://doi.org/10.1016/j.dyepig.2020.108376
DYPI 108376
Reference:
To appear in: Dyes and Pigments
Received Date: 26 December 2019
Revised Date: 11 March 2020
Accepted Date: 17 March 2020
Please cite this article as: Gao P, Jiang H, Chen W, Cui Z, An intramolecular Mannich type reaction of
ortho-amino aromatic azo dye and its detection effect for formaldehyde, Dyes and Pigments (2020), doi:
https://doi.org/10.1016/j.dyepig.2020.108376.
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CRediT author statement
Chen weiguo: Conceptualization, Methodology, Writing - Review & Editing, Project
administration
Cui zhihua: Conceptualization, Methodology, Writing - Review & Editing, Project
administration
Jiang hua: Methodology, Formal analysis
Gao pu: Investigation, Writing - Original Draft , Formal analysis, Writing- Reviewing
and Editing

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An intramolecular Mannich type reaction of ortho-amino aromatic azo dye and
its detection effect for formaldehyde
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Pu Gao a, b, Hua Jianga, c，Weiguo Chen*a, c and Zhihua Cui* *a, c
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aEngineering Research Center for Eco-Dyeing & Finishing of Textiles, Ministry of Education, Zhejiang Sci-Tech University,
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Hangzhou 310018, China
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bNanhu college, jiaxing university, Jiaxing 314001, China
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cKey Laboratory of Advanced Textile Materials and Manufacturing Technology, Ministry of Education of China, Hangzhou 310018,
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China
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*Corresponding author
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**Corresponding author
Email address:wgchen_62@126.com(W. Chen) and zhhcui@zstu.edu.cn (Z. Cui)
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Abstract: A series of aromatic primary amine dyes were designed and applied for the modification
of silk fibroin with Mannich-type reaction in the previous studies. Most aromatic primary amine dyes
achieved the expected results with the participation of formaldehyde. However, those azo dyes with
ortho-amino aromatic structure lost their colour and did not have any modification on silk fibroin
under the same conditions of treatment. Therefore, the decolorization phenomenon of ortho-aniline
aromatic azo dyes was analyzed using infrared, mass spectrometry, NMR and Gaussian computation
and the results indicated that an intramolecular Mannich reaction occurred between ortho-amino
aromatic azo dyes and formaldehyde. In addition, a secondary effect of reduction of dyes by
formaldehyde took place, in which the conjugated chromogenic system of the dye molecules was
destroyed, producing benzimidazole, o-phenylenediamine and nitrogen-containing heterocyclic
substances with polymethyl substitution. This decolorization of dye was studied as a detection or
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monitoring method for formaldehyde, and it showed advantages of sensitive response, clear visual
indication, and no volatilization of organic solvent.
Keywords: Intramolecular; Mannich type reaction; Decoloration; Ortho-amino aromatic azo dye;
Detection; Formaldehyde.
1. Introduction
In the 19th century, the Mannich reaction (named after Carl Mannich) was discovered [1]. Due to
the numerous forms of Mannich reaction, it has been developed into an important synthetic reaction
and is widely used in the synthesis and modification of drugs, dyes and natural compounds [2].
Mannich reactions include intermolecular and intramolecular reactions, with intramolecular reactions
being more widely used.
Formaldehyde plays a vital role in the Mannich reaction，but is a cytotoxic substance [3, 4] and
an environmental hazard. The world health organization's international agency for research on cancer
(IARC) has listed formaldehyde as a grade No. 1 carcinogen [5]. It can readily penetrate the human
skin and mucous membrane and cause industrial diseases. However, the application of formaldehyde
has been involved in all aspects of life, especially in textiles [6], cosmetics [7], paints [8, 9], and
foods [10-12]. Because formaldehyde is colorless, tasteless and volatile, improving the sensitivity for
detection and monitoring of formaldehyde has been the focus of research in this field. At present,
spectrophotometry and chromatography are the applied methods for detection of formaldehyde, and
the spectrophotometer method is relatively fast, convenient and more widely used. In this method,
the indicators often used include acetylacetone, heptamethine cyanine derivative (HCy1) [13],
AIE-based fluorescent probe [14], chromotropic acid, AHMT, phloroglucinol, and phenylhydrazine
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hydrochloride [15]. However, there are also some problems. For example, even though acetylacetone
is the most widely used, its sensitivity is rather low, and the method is also susceptible to
interference from propanal, butyl aldehyde and acrolein [16, 17]. It can also be decomposed at room
temperature. It is important to look for a more sensitive and reliable formaldehyde indicator.
The modification of proteins by aromatic primary amines based on the Mannich reaction is an
effective chemical modification method [18, 19]. In our previous studies [20, 21], a series of
aromatic primary amine compounds containing a chromophore (aromatic primary amine dyes) has
been designed and used for the modification of silk fibroin with the participation of formaldehyde
based on the Mannich reaction. It clearly showed that the reaction occured due to the development of
fast color visibly on silk. In the meantime, another phenomenon was observed. The ortho-amino
aromatic azo dyes did not achieve the same result on silk fibroin as other aromatic primary amine
dyes did. It was found that when an ortho-amino aromatic azo dye was added to formaldehyde, a
sensitive decoloration phenomenon occurred. This paper will discuss the reasons of the decoloration
of ortho-amino aromatic azo dyes by formaldehyde, and also the possibility for detection of
formaldehyde in water.
2. Experimental section
2.1. Synthesis of ortho-amino aromatic azo dyes
Ortho-nitroaniline was used as the heavy nitrogen diazonium component, and 1- (4-sulfonic acid
phenyl) -3-methyl-5-pyrazolone was used as the coupling component. Ortho-amino aromatic azo dye
(here in after referred to as dye D1) was prepared by coupling reaction and reduction reaction
successively. The synthetic routes of dyes D2 and D3 was the same as that of D1，but the coupling
component of D2 and D3 was sodium 2-naphthol-3,6-disulfonate and sodium
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2-naphthol-6-disulfonate respectively. The synthesis reaction is shown in Fig.1.
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D1: λmax434 nm. FTIR (ATR, cm-1): 3385 (NH2), 1164, 1028 (-SO3H). H NMR (400 MHz,
DMSO-d6) δ 7.94 (d, J = 7.3 Hz, 2H), 7.66 (d, J = 7.0 Hz, 2H), 7.46 (d, J = 7.4 Hz, 1H), 7.02 (t, J =
7.9 Hz, 1H), 6.91 (d, J = 7.5 Hz, 1H), 6.78 (s, 1H), 5.35 (s, 2H), 2.32 (s, 3H). ESI MS (m/z, %):
372.2 (M-H,100).
D2：λmax519 nm. FTIR (ATR, cm-1): 3436 (NH2), 3238, 3073, 1624, 1487 (Ar-H), 1184, 1036
(-SO3H). 1H NMR (400 MHz, DMSO-d6) δ 8.45 (d, J = 7.8 Hz, 1H), 8.28 (s, 1H), 8.06 (s, 1H), 7.96
(d, J = 8.0 Hz, 1H), 7.81 (t, J = 8.0 Hz, 1H), 7.23 (t, J = 8.0 Hz, 1H), 7.01 (d, 7.91 Hz , 1H), 6.82 (s,
1H). ESI MS (m/z, %): 210.50 (M-2H), 100).
D3：λmax500 nm. FTIR (ATR, cm-1): 3474, 3353 (NH2), 3040, 2908, 1591, 1503 (Ar-H), 1157,
1024 (-SO3H). 1H NMR (400 MHz, DMSO-d6) δ 14.57 (s, 1H), 8.50 (d, J = 8.7 Hz, 1H), 8.15 (s, 1H),
8.06 (d, J = 9.0 Hz, 1H), 7.86 (d, J = 8.7 Hz, 1H), 7.79 (d, J = 8.1 Hz, 1H), 7.27 (d, J = 7.1 Hz, 1H),
7.22 (d, J = 9.1 Hz, 1H), 7.07 (d, J = 9.5 Hz, 1H), 6.88 (t, J = 7.4 Hz, 1H). ESI MS (m/z, %): 342.08
(M-H, 100).
O2N
H2N
O2N
-Cl+N2
HCl, NaNO2
0-5
CH3
CH3
O2N
-Cl+N2
O2N
N
N
N
pH 7-8
0-5
N
N
N
O
O
HO3S
HO3S
CH3
CH3
O2N
N
N
N
H2N
N
N
N
N
N
Na2S
O
H
O
NaHCO3
HO3S
HO3S
D1
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4

O2N
N
H2N
N
N
N
O2N
-Cl+N2
OH
pH 9-10
0-5
OH
OH
SO3H
Na2S
+
NaHCO3
HO3S
SO3H
HO3S
SO3H
HO3S
D2
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O2N
N
N
O2N
-Cl+N2
OH
pH 9-10
0-5
OH
+
HO3S
HO3S
H2N
H2N
N
H
N
N
N
Na2S
O
OH
NaHCO3
HO3S
HO3S
D3
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Fig. 1. Synthesis route of ortho-amino aromatic azo dyes D1, D2 and D3
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2.2. Separation of decolorization reaction products
200 mg pure dye D1 was dissolved in 2000 mL distilled water, kept at constant temperature in 30℃
water bath for 5 minutes, the pH value of the solution was adjusted to 4.5 with acetic acid, then the
absorbance of the solution was measured and recorded. Then 10% formaldehyde solution was added
into the solution. When the color of the solution was close to colorless, the formaldehyde solution
was added drop by drop and waited for 3 minutes after each drop. The solution was evaporated at 60℃
in a low pressure rotary evaporator, and the dried colorless solid material obtained was sealed under
nitrogen for test of FT-IR and ESI-MS. The solid material was dissolved with 100 mL anhydrous
ethanol to extract the alcohol soluble components, the supernatant was taken away and dried at 60℃
in the low pressure rotary evaporator to obtain the solid samples for measurement of 1H NMR.
2.3. FT-IR test
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An appropriate amount of dye D1 and its decolorization product were taken, and ATR test was
carried out by Nicolet 5700 Fourier transform infrared spectrum analyzer, Thermo Nicolet
Corporation, USA, with 32 scanning times and resolution of 4 cm-1.
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2.4. ESI-MS test
An adequate amount of sample was configured to the concentration of 500 ppm solution with
distilled water. A suitable amount of solution was filtered through water molecular sieve, and placed
into a sample bottle. An LCQ Fleet liquid ion trap mass spectrometer was used to determine accurate
molecular ion peaks of the sample.
2.5.1H NMR test
The alcohol-soluble solid obtained in 2.2 was dissolved in 0.6 mL deuterium DMSO, and the
product was tested by FT-NMR Digital NMR spectrometer.
2.6. Detection test for formaldehyde using ortho-amino aromatic azo dye
2.6.1. Absorbance - concentration standard work curve drawing
A dye D1 solution of 0.001 mol/L was prepared with distilled water. The solution was diluted to
10, 100, 1000, 2000, 5000, 6000, 8000 times and the absorbance was measured by UV-visible
spectrophotometer at wavelength of 435 nm as its λmax, the standard working curve is drawn as Fig.2.
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10
8
6
4
2
0
0
2
4
6
8
10
Concentration (mol/L)*10-6
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Fig. 2. Standard working curve of dye D1
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2.6.2. UV-Vis spectroscopy characterization experiment of decolorization phenomenon
Distilled water was used to prepare 50 mL formaldehyde solution with a concentration of 0.001
mol/L at a temperature of 30℃. 0.01 mL of dye D1 solution with a concentration of 0.001 mol/L was
added. The absorbance of the solution was measured at 0, 5, 15, 30 and 60 min respectively, and then
a curve was drawn.
2.6.3. Experiment of the effect of temperature on decolorization phenomenon
0.3 mL of 0.002 mol/L indicator dye D1 solution was added into the reagent tube, and then 10 mL
0.002 mol/L formaldehyde solution was added. The tube was shaken well and put into a 30oC water
bath for 3 min and absorbance was measured repeatedly. The temperature was raised from 60℃ to
90℃, and the absorbance was measured at the same time interval.
2.6.4. Experiment of the effect of pH value on decolorization phenomenon
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10 mL 0.002 mol/L formaldehyde solution was placed into 6 test tubes, their pH values were
adjusted to 2.0, 3.0, 4.0, 7.0, 10.0, 12.0 using hydrochloric acid and sodium hydroxide, and the tubes
were placed in a 30℃ water bath at a constant temperature for 3min. 0.3 mL of 0.002 mol/L dye D1
solution was added to each tube. The tubes were shaken well, and the solutions allowed to stand for 2
min, and then tested every 3 min until the absorbance no longer changed.
2.6.5. Experiment to determine the working curve of indicator application
0.002 mol/L of dye D1 solution was prepared and 0.02 mL was added to a series of formaldehyde
standard solutions with different concentrations. The mixture was then reacted at 90℃ for 5 min,
and the absorbance of the mixture after the reaction was measured, so as to obtain the decreased
value of the absorbance of the mixture.
3. Results and discussion
3.1. Decolorization of ortho-amino aromatic azo dyes with formaldehyde
As described in 2.6.2, 0.01 mL of dye D1, D2 or D3 solution with a concentration of 0.001 mol/L
were added to 50mL 0.001 mol/L formaldehyde solution, the UV-visible spectrum of decolorization
phenomenon is shown in Fig. 3. We can see from A in Fig. 3 that the absorbance of visible light (435
nm) decreased with extension of reaction time, while the absorptivity of UV light increased at 345
nm and 250-270 nm. The phenomenon indicates that in the process of the reaction, the dye D1 and
formaldehyde gradually reacted with each other to form new colourless substances. The dye's
original color gradually faded, and the reaction products formed gradually increased, making its
characteristic absorption peak in the ultraviolet region. After 60 min, the visible absorption peak
declined to zero and the solution became colorless. It indicated that the reaction caused
decolorization and complete color fading.
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In addition to the decolorization of dye D1, the other two ortho-amino aromatic azo dyes D2 (440 nm)
(B in Fig.3) and D3 (517 nm) (C in Fig.3) also clearly showed the phenomenon of decoloration.
These results reveal that ortho-amino aromatic azo dyes under the appropriate conditions can react
with formaldehyde to form a colorless substance and the chromophore of the original dye will no
longer exist.
0.6
A
1--Dye D1 solution
0.5
0.4
0.3
0.2
0.1
0.0
-0.1
2--5 min
3--15 min
4--30 min
5--60 min
1
2
3
4
5
300
400
500
600
Wavelength/nm
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155
B
Dye D2 solution
Dye D2 solution with formaldehyde
200
300
400
500
600
700
Wavelength
Wavelength/nm
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157
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C
Dye D3 solution
Dye D3 solution with formaldehyde
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300
400
500
600
700
Wav length
Wavelength/nm
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Where A, B and C respectively represent the UV-visible spectrum of dye D1, dye D2 and dye D3, showing the
decoloration phenomenon. Curve 1 represents the UV-visible spectrum of the dye D1 solution，and curve 2, 3, 4, 5
represents the UV-visible spectrum of dye D1 reacted with formaldehyde for a certain time. The concentration of
formaldehyde solution is 0.001 mol/L.
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Fig. 3. The UV-visible spectrum of decoloration phenomena of ortho-amino aromatic azo dyes
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3.2. Study on the decolorization reaction mechanism of ortho-amino aromatic azo dyes
Fig.4 shows the molecular structure of dye D1, which is a pyrazolinone structure azo dye.
Azo-hydrazone isomerism exists in dye D1, which is usually in the form of the hydrazine [22-24].
Fig. 5 shows the infrared spectrum of dye D1 and its decolorization products. It can be seen from the
figure that 3319 cm-1 in the spectrum of dye D1 is the stretching vibration peak of the primary amino
group. 1645 cm-1 is the telescopic vibration absorption peak of pyrazolone carbonyl (-C=O), and
1544 cm-1, 1497 cm-1 are the vibration absorption peaks of the benzene ring skeleton hydrogen
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(-Ar-H). The weak peak around 2930 cm-1 is the telescopic vibration absorption signal of methyl.
1127 cm-1, 1039 cm-1 are the antisymmetric and symmetric telescopic vibration absorption peaks of
dye sulfonic acid group (-SO3H) respectively, and 1645 cm-1 is the absorption peak of -C=O.
CH3
CH3
H2N
N
N
N
H2N
N
N
N
N
N
O
O
H
HO3S
HO3S
Azo, tiny amount
Hydrazone, overwhelming amount
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Fig. 4. The Azo-Hydrazone tautomerism of ortho-amino aromatic azo dyes D1
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In the infrared spectrum of the decolorization products, the 3426 cm-1 and 3359 cm-1 are the
stretching vibrations of the amino group. 1126 cm-1 is the anti-symmetric and symmetric telescopic
vibration absorption peak of -SO3H. A narrow but strong absorption peak appeared near 2986 cm-1,
2922 cm-1, and the 1388 cm-1 and 1470 cm-1 which could be attributed to the stretching vibration of
-CH3. Compared with the spectrum of dye D1 before decolorization, the stretching vibration of -CH3
was significantly enhanced, indicating that one or more new -CH3 was possibly generated in the
reaction products. The peak at 1639 cm-1 is -N=C absorption peak [25]. Since -C=O absorption
signal is merged, the peak becomes broader.
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90
2930
80
1645
1639
1127
70
1039
3426
3359
1470
1388
2986
3319
60
1037
50
2922
40
Dye D1
Decolorization products
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1126
20
0
500
1000 1500 2000 2500 3000 3500 4000 4500
Wavenumber (cm-1)
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Fig. 5. Infrared spectrum of dye D and decolorization products
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Fig. 6 shows the mass spectrogram of dye D1 and its decolorization products. The strong peak 372
in the negative mode in A in Fig. 6 is the [M-H]- of the dye. However, the peak 372 was not observed
in the decoloration product in the negative mode of B in Fig. 6, and the [M-H]- of the dye completely
disappeared, while strong peaks appeared at 107, 117, 171, 285 (286), and a strong peak of 119
appeared in the positive mode of B in Fig. 6. These are the signal peaks of the new substances
generated by the reaction between the dye and formaldehyde. These results revealed that new
substances were definitely formed.
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A is the mass spectrogram of dye D1; B is the mass spectrogram of decolorization products; C is the mass
spectrogram of dye D1 solution and its decolorization products solution placed at room temperature for 60 days.
Fig. 6. ESI-MS before and after decolorization of dye D1
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The dye D1 solution before decolorization and the decolorization product solution were placed at
room temperature for 60 days, and their mass spectrograms were tested further. The results are
shown in C in Fig. 6. The dye still has 372 strong peak [M-H]- after being placed in aqueous solution
for 60 days. It can be preliminarily concluded that the dye has good storage stability in water. The
spectrum of the decolorization products shown in B in Fig. 6 is significantly different from the mass
spectrogram of dye solution and its decolorization products solution placed at room temperature for
60 days shown in C in Fig. 6. The peaks of 107, 117, 171, 285 (286) found in B in Fig. 6 all
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disappeared in C in Fig. 6, and a strong peak of 295.95 appeared, indicating that after longtime
storage, decolorization products further changed to form new substances.
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Fig. 7. 1H NMR of alcohol-soluble components of the decolorization products
Table 1 1H NMR analysis of alcohol-soluble components of the decolorization products
Proton number Chemical shift (ppm) Assignment
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1
2
3
4
7.18~7.20(m)
7.59(s)
Ar-H
Ar-H
N-H
C-H
12.42(s)
8.21(s)
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In order to further clarify the reaction products, the mixed products were dissolved with ethanol,
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and the ethanol soluble substances were tested using H NMR. The results are shown in Fig. 7 and
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Table 1. It can be inferred from the analysis results that the ethanol-soluble component in the
decoloration products is benzimidazole [26, 27].
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Fig. 8. The Mulliken charges of dye D1
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In the first step of the Mannich reaction mechanism, the protonated formaldehyde molecule is an
electrophilic center which is easily attacked by electron-rich N atoms. As the dye D1 molecule
contains a variety of nitrogen atoms, it is important to determine the electronegativity of each N atom,
which provides an important reference for determining the reaction site of Mannich reaction, and
thus for deriving its reaction mechanism. Therefore, we used Gaussian computation software to
calculate the Mulliken charges of each N atom in the dye D1 molecule by using BYLYP/3-21G. The
computation results are shown in Fig. 8. By comparison, it can be seen that the electron cloud of No.
(1) N atom has the highest density and exists in the form of a primary amine. In the Mannich reaction
process, it is the first N atom to react with formaldehyde to form a Schiff base, acting as the Mannich
reactive center, namely the initiation site of the decolorization reaction. Secondly, when the resulting
Schiff base is protonated, it prefers to attack N atoms with lower electron density. Although the
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electron cloud of No. (4) N atom is relatively dense, it is too far away from No. (1) N atom to be a
target for attack by proton Schiff base. Therefore, the No. (2) N atom is likely to participate in the
intramolecular Mannich reaction as the object most likely to be attacked by the protonated Schiff
base.
The decolorization reaction mechanism of ortho-amino aromatic azo dyes was deduced by a
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comprehensive analysis of the results of FT-IR spectrum, ESI-MS, H NMR and Gaussian software
computation. The most probable decolorization reaction includes two reaction processes (Fig. 9).
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Fig. 9. The possible mechanism of decolorization reaction
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In the reaction process 1, formaldehyde is first converted into an electrophilic reagent under the
catalysis of H+, and then binds to the No. (1) N atom of amine group with high electronegativity.
Then it is further dehydrated to form a protonated Schiff base, and then further attacks the adjacent
No. (2) N atom with lower electronegativity, resulting in the disconnection of No. (2) N and No. (3)
N. This reaction leads to the formation of the benzimidazole with molecular weight of 118, which
corresponds to the 117 [M-H]- in B in Fig. 6. For the other part of the pyrazolinone structure, when
the formaldehyde concentration is high or the reaction time is sufficient, formaldehyde plays the role
of a reducing agent to reduce the product into a structure where No. (2) N atom is a Schiff base.
Since the Schiff base formed at the end-position unconjugated system is not stable, it will be further
isomerized into the product with molecular weight of 297, corresponding to the 295.96 [M-H]-
product in C in Fig. 6.
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In reaction process 2, when the concentration is high or the reaction time is sufficient, the reduction
effect of formaldehyde would be greater. The No. (2) and No. (3) N-N bond can be directly reduced
and broken to form ortho-phenylenediamine, corresponding to the molecular weight of 107 in B in
Fig. 6. At the same time, the other product can continue the reaction under the effect of formaldehyde
reduction and finally form the reaction product with molecular weight of 297. In the whole reaction,
decolorization phenomenon occurs due to the fracture of the conjugated system.
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3.3. Effect of ortho-amino aromatic azo dye on formaldehyde detection
In order to explore the application of ortho-amino aromatic azo dye D1 for formaldehyde detection,
the influences of temperature and pH value on decolorization were discussed, and the working curve
of ortho-amino aromatic azo dye D1 in formaldehyde detection was drawn.
3.3.1. The effect of temperature on decoloration
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Fig.10 shows the effect of temperature on the decoloration rate. As can be seen from the figure, the
effect of temperature on decoloration reaction is obvious, the higher the temperature, the faster the
decoloration. The decoloration reaction can be completed in 3 minutes at 90 and 7 minutes at 60 .
Therefore, in order to ensure the sensitivity of detection, ortho-amino azo reagent is suitable for
higher temperatures.
0.35
30℃
60℃
0.30
0.25
0.20
0.15
0.10
0.05
0.00
90℃
0
5
10
15
20
25
30
35
40
t/min
278
279
Fig. 10. The effect of temperature on decoloration
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281
3.3.2. The effect of pH values on decoloration
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Fig. 11 shows the effect of pH value on the decoloration rate. It can be seen from the figure that
pH value has an obvious effect on the decoloration rate. When pH value is 10.0 or 2.0, the
decoloration occurs quite slowly, both strong acid and strong alkaline conditions do not assist
decolorization. When the pH value is 4.0, the fastest decoloration can be completed within 30 min.
This is because the Mannich reaction is catalyzed by protons, and the proper concentration of H+ in
the solution is essential for the reaction to occur smoothly. The first step of the Mannich reaction is
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289
290
291
the protonation of the aldehyde group. Due to the lack of H+ in alkaline conditions which doesn’t
allow formaldehyde to be protonated, the Mannich reaction cannot occur quickly. On the other hand,
in the strong acidic conditions, the amino group has a certain alkalinity and is easy to be neutralized
by H+ to become an ammonium salt, thus it is no longer an electrophilic center.
0.35
0.30
0.25
pH=2.0
pH=4.0
pH=6.0
pH=8.0
pH=10.0
0.20
0.15
0.10
0.05
0.00
0
10
20
30
40
t/min
50
60
70
80
292
293
294
Fig. 11. The effect of pH values on decoloration
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297
298
299
3.3.3. Application curve of ortho-amino aromatic azo dye D1 to formaldehydedetection
In order to explore the relationship between decolorization and formaldehyde concentration, the
natural logarithm of formaldehyde concentration (LnC) was taken as the horizontal coordinate, and
the decline in value of absorbance (dA) of mixed solution after dropping dye D1 was taken as the
vertical coordinate, and the curve was drawn as shown in Fig.12.
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C is the concentration of formaldehyde mg/mL, A is the absorbance decline value of the mixed solution after
dropping ortho-amino aromatic azo dye D1 into formaldehyde solution.
Fig. 12. The relationship between the decolorization of ortho-amino aromatic azo dye D1 and the formaldehyde
concentration
302
303
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305
306
307
308
309
310
311
312
313
314
As can be seen from the Fig. 12, dA was used for linear fitting of LnC data, and the fitting degree
was 98.8%. The good linear relationship indicated that the reaction between dye D1 and
formaldehyde followed a certain rule, and the concentration of formaldehyde could be characterized
by the reduced absorbance of the mixed solution. Moreover, when the horizontal coordinate value is
about 9.4, the linear relationship is good, that is, it is possible to detect the formaldehyde
(-9.4)
concentration higher than e
=0.123mg/L of the liquid, also the qualitative detection of
formaldehyde can still be achieved when the formaldehyde concentration is lower than this value.
And the limit of detection (LOD) of ortho-amino aromatic azo dye D1 decoloration calculated by 3δ
rule is 0.105mg/L.
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4. Conclusions
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323
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325
326
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328
329
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331
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In conclusion, the decolorization reaction mechanism between ortho-amino aromatic azo
dyes and formaldehyde has been clarified into two aspects: the intramolecular Mannich
reaction of dye reacted by formaldehyde, and the reduction effect on dye caused by
formaldehyde which destroys the conjugated chromogenic system formed by pyrazolone and
ortho-amino azo inside the dye molecules. The reaction finally produces benzimidazole,
o-phenylenediamine and nitrogen-containing heterocyclic substances with polymethyl
substitution. This decolorization reaction, with strong visibility and sensitivity, can be used
for detection or monitoring of formaldehyde, and the reaction is fastest and most sensitive at
90
℃ and pH 4.0. The working curve of this reaction in formaldehyde indication is linear and
the detection limit is 0.123 mg/L. The application of ortho-amino aromatic azo dye in
accurate detection or monitoring of formaldehyde has the advantages of a sensitive response,
clear visual indication, no volatilization of organic solvent, and stable storage of indicator
solution.
Acknowledgements
This work was supported by National Natural Science Foundation of China (Nos. 51673176,
21808210), Zhejiang Provincial Natural Science Foundation of China (LY16B060006), and Public
Welfare Technology Research Project of Zhejiang Province (LGG18B060003), Key project of nanhu
college of jiaxing university（N41472001-42）.
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24

Highlights
1. A noval and sensitive decolorization phenomenon of ortho-aniline aromatic azo
dyes and was reported.
2. And the decolorization reaction mechanism between ortho-amino aromatic azo
dyes and formaldehyde has been clarified into two aspects: the intramolecular
Mannich reaction of dye reacted by formaldehyde, and the reduction effect on dye
caused by formaldehyde which destroys the conjugated chromogenic system formed
by pyrazolone and ortho-amino azo inside the dye molecules.
3. This decolorization reaction, with strong visibility and sensitivity, can be used for
detection or monitoring of formaldehyde.

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: