Microdroplet synthesis of azo compounds with simple microfluidics-based pH control

Article abstract of DOI:10.1039/d0ra06344d

Conventional solution-phase synthesis of azo compounds is complicated by the need for precise pH and temperature control, high concentrations of pH control reagents, and by-product removal. In this work, we exploited the advantages of microdroplet chemist

Full text of DOI:10.1039/d0ra06344d

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Microdroplet synthesis of azo compounds with
simple microfluidics-based pH control
Cite this: RSC Adv., 2020, 10, 38900
a
Shunsuke Sawai,b Shohei Hattori,b Yoshito Nozaki,a
*
Daiki Tanaka,
a
Dong Hyun Yoon,
and Shuichi Shojib
Hiroyuki Fujita,d Tetsushi Sekiguchi,a Takashiro Akitsuc
Conventional solution-phase synthesis of azo compounds is complicated by the need for precise pH and
temperature control, high concentrations of pH control reagents, and by-product removal. In this work,
we exploited the advantages of microdroplet chemistry to realize the simple and highly efficient
synthesis of an azo compound using microfluidics-based pH control. Owing to the small size of
microdroplets, heat exchange between
a microdroplet and its environment is extremely fast.
Furthermore, chemical reactions in microdroplets occur rapidly due to the short diffusion distance and
vortex flow. Formation of the azo compound reached completion in less than 3 s at room temperature,
compared with 1 h at 0 ꢀC under conventional conditions. pH control was simple, and the pH control
reagent concentration could be reduced to less than one-tenth of that used in the conventional
method. No by-products were generated, and thus this procedure did not require a recrystallization
step. The time course of the chemical reaction was elucidated by observing the growth of azo
compound microcrystals in droplets at various locations along the channel corresponding to different
mixing times.
Received 21st July 2020
Accepted 15th October 2020
DOI: 10.1039/d0ra06344d
rsc.li/rsc-advances
to product degradation, so the pH should be returned to
neutral. Zhao et al. reported the successful one-pot solution-
1. Introduction
phase preparation of various azo compounds by varying the
synthesis time and temperature.3 They showed that prolonged
stirring and strict temperature control were necessary for the
synthesis of azo compounds. Einaga et al. systematically
investigated organic/inorganic hybrid materials composed of
metal Schiff base complexes and azo compounds in poly(methyl
methacrylate) lms as non-crystalline solids4 that could be
made to regularly align upon light irradiation.5 They succeeded
in photoisomerization and structural modication of azo
compounds by attaching an azo group to a palladium complex.
However, these conventional solution-phase synthetic methods
involved a complicated procedure using a metal catalyst and
strict control of the atmosphere and temperature.
Azo compounds are versatile materials used in anticancer
drugs, batteries, and numerous other applications. For
example, Ganguly et al. reported that the copper complex of an
azo compound exhibited anticancer activity by strongly binding
to DNA,1 while Luo et al. used an azo compound to fabricate the
organic electrode of a lithium-ion battery.2 Organic electrode
materials are promising as materials for batteries but have the
problem that they dissolve in the liquid electrolyte, suppressing
battery capacity. The use of an azo compound electrode miti-
gates this problem. Although azo compounds have an array of
applications in various elds, their synthesis remains chal-
lenging and is still an active area of research.
Azo compounds are conventionally synthesized via
a complicated solution-based procedure. For instance, the
We previously established
a simple method for azo
ꢀ
compound synthesis using a Y-shaped microuidic device
under laminar ow.6 Although this method was superior to the
conventional method in terms of reaction time and tempera-
ture, the resulting azo compound clogged the channel. There-
fore, we herein describe a new method for the synthesis of azo
compounds in which all of the complicated synthetic steps
occur within microdroplets. The azo compound synthesized
inside the microdroplets ows to the outlet without clogging the
channel. The reagent introduced into the microdroplets
diffuses instantaneously throughout, which enables efficient
chemical reaction.7,8
temperature should be maintained at 0 C to remove the heat
generated by the reaction. Furthermore, the diazotization step
is typically conducted in highly concentrated aqueous hydro-
chloric acid solution to promote the chemical reaction.
However, high concentrations of acidic or alkaline reagents lead
aResearch Organization for Nano
& Life Innovation, Waseda University, Tokyo
162-0041, Japan. E-mail: d.tanaka@ruri.waseda.jp
bFaculty of Science and Engineering, Waseda University, Tokyo, 169-8555, Japan
cDepartment of Chemistry, Faculty of Science, Tokyo University of Science, Tokyo 162-
8601, Japan
dCanon Medical Systems Corporation, Otawara, Tochigi 324-8550, Japan
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Microdroplet-mediated chemical synthesis has been studied within microdroplets. Fig. 1b presents an overview of the
primarily in the eld of organic chemistry.9–12 Song et al. microuidic experiment. The individual microdroplets fused
compared the mixing of reagents during chemical reactions inside the microuidic device, permitting the synthetic reaction
under laminar ow and inside microdroplets.13 Using a simpli- to proceed.
ed diffusion model, Mortensen and Williams reported that
mixing occurs in less than a millisecond, and the contribution
of turbulence, estimated from times of coalescing ballistic
microdroplets, suggests that complete mixing occurs within
a few microseconds.14 Ursuegui et al. reported a microdroplet
surface engineering strategy involving strain-promoted alkyne–
azide cycloaddition, in which they designed and synthesized an
azide PFPE–PEG based uorosurfactant prone to react with
strained alkyne derivatives via a copper-free click reaction.15 The
use of microdroplets leads to more efficient mixing compared
with laminar ow and is advantageous for chemical
synthesis.16–19 Theberge et al. integrated two enabling technol-
ogies, namely, droplet-based microuidics and uorous
biphasic catalysis, to create controlled catalytic interfaces for
organic synthesis in ow.20 Although organic synthesis using
microdroplets can provide superior results in terms of reaction
time and temperature, no successful examples have yet been
reported for reactions requiring strict pH control. In this study,
we synthesized complex azo compounds using simple
microuidics-based pH control.
2.3. Device design and microuidic experiments
Fig. 1c shows the design of the microdroplet-generating device
used in this study. The device was constructed from poly-
dimethylsiloxane (PDMS) with a channel width and depth of
200 and 100 mm, respectively.21 Solutions of the reagents were
introduced via syringe using a syringe pump. Fig. 2 shows the
microuidic device used in this study. Microuidic devices of
the same design are placed one above the other to switch when
there is a problem.
The azo compound was synthesized using aniline (250 mmol
Lꢁ1, reagent A), sodium nitrite (250 mmol Lꢁ1, reagent B), and o-
vanillin (250 mmol Lꢁ1, reagent C). The aniline and o-vanillin
were dissolved in aqueous HCl solution (500 mmol Lꢁ1) and
aqueous NaOH solution (500 mmol Lꢁ1), respectively. The pH
and reagent concentration were varied by altering the ow rate
to examine their effect on the chemical reaction. The measured
pH values were 3, 7, and 12.
The synthesized azo compound was isolated by ltration and
analysed by infrared (IR) spectroscopy (FT/IR-6200, JASCO), fast
atom bombardment mass spectrometry (FAB-MS; JMS-BU25,
2. Experimental
2.1. Materials
Chemicals and reagents were of the highest grade commercially
available (Tokyo Chemical Industry or Kanto Chemical) and
were used as received without further purication.
2.2. Synthesis concept and scheme
Fig. 1a shows the two-step reaction scheme used to synthesize
an azo compound. The azo compound was synthesized from
three reagents, namely, aniline, sodium nitrite, and o-vanillin;
the conventional synthetic method using these reagents
requires precise pH control. Each of the reagents was conned Fig. 2 Microfluidic device used in this experiment.
Fig. 1 Synthesis of azo compounds: (a) synthetic scheme, (b) outline of the microfluidic experiment, and (c) design of microfluidic device.
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Table 1 Comparison of the synthesis conditions
Beaker
0–5 ꢀ
Microdroplet
Synthesis temperature
Synthesis time
Concentration of pH control reagents
C
Room temperature
3 s
3600 s
5.6 mol Lꢁ1
0.5 mol Lꢁ1
JEOL), and 1H and 13C nuclear magnetic resonance (NMR) contained a peak with a mass-to-charge ratio (m/z) of 257.10,
spectroscopy (ECX500, JEOL).
which corresponds to the [M + H]+ ion of the expected product
(Fig. 4b). Fig. 4c and d show the 1H and 13C-NMR spectra,
respectively. The analytical results were consistent with those
expected for the desired azo compound.
3. Results and discussion
The azo compound shown in Fig. 1a, which typically requires
a complicated synthetic procedure, was synthesized inside
microdroplets. The proposed method has several advantages
over the conventional solution-phase method (Table 1). In
particular, control of the reaction temperature was unnecessary
and the reaction could be performed at room temperature (23
ꢀC) rather than 0 ꢀC, while the reaction time decreased from 1 h
to 3 s or less.
3.2. Reaction rate in microdroplets
The synthesis of the azo compound inside the microdroplets
reached completion in approximately 3 s. Furthermore, the
concentration of the pH control reagents could be greatly
reduced. It is considered that the diffusion distance of the
chemical species inside the microdroplets was very short and
these species diffused throughout the microdroplets
instantaneously.
Fig. 3 shows microscopy images of the microuidic experi-
ment. Crystals of the azo compound formed immediately aer
the microdroplets fused. The chemical reaction inside the
microdroplets reached completion in 1.5–3 s. The synthesis
conditions were signicantly different from those used in the
conventional solution-phase method in a beaker. In the
microdroplet method, the concentration of the pH control
The diffusion time of a chemical species is proportional to
the square of the diffusion distance, as given by eqn (1):
d2
tf
(1)
D
reagents (HCl and NaOH) could be decreased to 0.50 mol Lꢁ1
,
where t is the diffusion time, d is the diffusion distance, and D is
the diffusion coefficient. The high diffusion efficiency in the
microscale reaction region therefore allowed the concentration
of the pH control reagents to be greatly reduced.
The diffusion time for the microdroplets employed in this
study was calculated to be 3.125 s using eqn (2):
which is less than one-tenth of that used in the conventional
method (5.62 mol Lꢁ1).
3.1 Product analysis
Fig. 4 shows the analytical results for the obtained azo
compound. A peak corresponding to N]N (1580 cmꢁ1) was
observed in the IR spectrum (Fig. 4a). The FAB-MS spectrum
d ¼ (Dt)0.5
(2)
The mutual diffusion coefficient D of hydrochloric acid and
water at 0.5 mol Lꢁ1 is approximately 3.2 ꢂ 10ꢁ9 m2 sꢁ1, and the
diffusion length d is 100 mm. In the microdroplet experiments,
the reaction time for the synthesis of the azo compound was
approximately 3 s, which is close to this theoretical value.
However, other factors in addition to diffusion also play a role in
actual experiments, such as the generation of eddy currents in
the droplets.
The microdroplet method allowed the concentration of the
pH control reagents to be drastically reduced from 5.6 mol Lꢁ1
to 0.5 mol Lꢁ1. Theoretically, the diazotization reaction can
proceed with 0.5 mol of HCl (H+) for 250 mmol of aniline
(Fig. 5). However, 5.6 mol Lꢁ1 of HCl (H+) is typically used in
conventional solution-phase synthesis, where the diffusion
distance is long and the reaction does not proceed unless at
least a tenfold excess of HCl is present.
On the other hand, in the microdroplet method, the diazo-
tization reaction proceeded at a HCl concentration of only
0.5 mol Lꢁ1. These results indicate that the chemical species
Fig. 3 Images of the microfluidic experiment (scale bar: 200 mm).
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Fig. 4 Characterization of the isolated azo compound: (a) IR spectrum, (b) FAB-MS spectrum, (c) 1H-NMR spectrum, and (d) 13C-NMR spectrum.
operations to be performed with cooling over ice. In contrast,
with the microdroplet method, it was possible to perform the
synthesis at room temperature (23 ꢀC). Microdroplets have
a very large surface area per unit volume, precise heat control
can be easily achieved. In particular, for a synthetic scheme
involving heat generation as in the current case, it is considered
that the heat generated by the chemical reaction rapidly diffuses
and dissipates to the surroundings. The microdroplet method
thus enabled efficient removal of reaction heat, thereby sup-
pressing side reactions.
3.4. Microscopic reaction observation
In this study, the pH was controlled by the ow rates of the pH
Fig. 5 Mechanism of the diazotization reaction.
control reagents (HCl and NaOH), as summarized in Table 2.
The concentration of the pH control reagents, and therefore the
can rapidly diffuse inside the microdroplets and the chemical
reaction can proceed in an optimal state.
Table 2 Relationship between pH and flow rates of the pH control
reagents
When two liquids are mixed inside a microdroplet, a vortex is
generated inside, thus promoting rapid mixing of the chemical
species and increasing the efficiency of the chemical reaction
beyond that possible via natural diffusion alone.
Flow rate [mL minꢁ1
]
Reagent
pH ¼ 3
PH ¼ 7
pH ¼ 12
Carrier uid 2.0
HCl
NaOH
2.0
2.0
3.3. Efficiency of temperature control
1.2 (1.0 mol Lꢁ1
0.6 (0.5 mol Lꢁ1
)
)
0.6 (0.5 mol Lꢁ1
0.6 (0.5 mol Lꢁ1
)
)
0.2 (0.2 mol Lꢁ1
0.6 (0.5 mol Lꢁ1
)
)
Because the formation of an azo compound is an exothermic
reaction, the conventional method requires the synthetic
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rate, and synthetic experiments can be performed under various
conditions.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
This work was partially supported by Canon Medical Systems
Corporation and a Grant-in-Aid for Basic Scientic Research (A)
(No. 20H00336) from the Japanese Ministry of Education,
Culture, Sports, Science and Technology. The authors would
also like to thank the MEXT Nanotechnology Platform Support
Project of Waseda University.
Fig. 6 Microscopy images of the microdroplets formed at various pH
values (scale bar: 100 mm).
References
1 D. Ganguly, C. K. Jain, R. C. Santra, S. Roychoudhury,
H. K. Majumder, T. K. Mondal and S. Das, ChemistrySelect,
2017, 2, 2044.
2 C. Luo, X. Ji, J. Chen, K. J. Gaskell, X. He, Y. Liang, J. Jiang
and C. Wang, Angew. Chem., Int. Ed., 2018, 130, 8703.
3 R. Zhao, C. Tan, Y. Xie, C. Gao, H. Liu and Y. Jiang,
Tetrahedron Lett., 2011, 52, 3805.
4 Y. Einaga, R. Mikami, T. Akitsu and G. Li, Thin Solid Films,
2005, 493, 230.
5 Y. Aritake, T. Takanashi, A. Yamazaki and T. Akitsu,
Polyhedron, 2011, 30, 886.
reaction conditions, could be easily controlled by varying the
ow rates.
Fig. 6 shows microscopy images of the microdroplets
synthesized at pH values of 3, 7, and 12. At pH 3, ne particles
formed immediately aer fusion of the droplets but dis-
appeared aer approximately 3 s. Immediately aer the
fusion of the droplets, it is considered that the pH changed to
7 in part of the droplet with formation of the azo compound,
but aer 3 s, the pH of the entire droplet became 3 and the
reaction did not proceed further. At pH 7, formation of the
azo compound proceeded steadily and the target compound
was successfully synthesized. At pH 12, an oily product was
observed. It is considered that replicative organisms and
excess reactants were produced because the pH was too high.
6 D. Tanaka, S. Sawai, D. H. Yoon, T. Sekiguchi, T. Akitsub and
S. Shoji, RSC Adv., 2017, 7, 39576.
7 H. Song, J. D. Tice and R. F. Ismagilov, Angew. Chem., Int. Ed.,
2003, 42, 767.
´
8 M. Wojnicki, M. Luty-Błocho, V. Hessel, E. Csapo, D. Ungor
and K. Fitzner, Micromachines, 2018, 9, 248.
9 Z. T. Cygan, J. T. Cabral, K. L. Beers and E. J. Amis, Langmuir,
2005, 21, 3629.
10 T. Fukuda, N. Funaki, T. Kurabayashi, M. Suzuki,
D. H. Yoon, A. Nakahara, T. Sekiguchi and S. Shoji, Sens.
Actuators, B, 2014, 203, 536.
4. Conclusion
The synthesis of azo compounds inside microdroplets has
numerous advantages over conventional solution-phase
synthesis. The microdroplet method permitted rapid 11 Y. Zheng, Z. Yu, R. M. Parker, Y. Wu, C. Abell and
synthesis of the desired azo compound at room temperature O. A. Scherman, Nat. Commun., 2014, 5, 5772.
(23 ꢀC), whereas conventional solution-phase synthesis in 12 E. K. Lumley, C. E. Dyer, N. Pamme and R. W. Boyle, Org.
a beaker typically requires cooling to 0 ꢀC over ice and a reac-
Lett., 2012, 14, 5724.
tion time that is 1000 times longer. Moreover, pH control 13 H. Song, J. D. Tice and R. F. Ismagilov, Angew. Chem., Int. Ed.,
could be conveniently accomplished and the concentration of 2003, 42, 767.
the pH control reagents could be reduced to one-tenth of that 14 D. N. Mortensen and E. R. Williams, Anal. Chem., 2014, 86,
typically required. All of the complex synthetic operations such 9315.
as pH adjustment occurred within the microdroplet. Further- 15 S. Ursuegui, M. Mosser and A. Wagner, RSC Adv., 2016, 6,
more, the microdroplet method efficiently afforded a high- 94942.
purity product without the need for recrystallization. Thus, 16 M. Faustini, J. Kim, G. Y. Jeong, J. Y. Kim, H. R. Moon,
the described technique permits the convenient synthesis of W. S. Ahn and D. P. Kim, J. Am. Chem. Soc., 2013, 135, 14619.
azo compounds that typically require complex synthetic 17 R. O. Grigoriev, M. F. Schatza and V. Sharmab, Lab Chip,
operations. 2006, 6, 1369.
Furthermore, the described microuidic device allows the 18 F. Sarrazin, L. Prat, N. Di Miceli, G. Cristobal, D. R. Linkc and
reagent concentration to be easily varied by controlling the ow
D. A. Weitz, Chem. Eng. Sci., 2007, 62, 1042.
38904 | RSC Adv., 2020, 10, 38900–38905
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Paper
RSC Advances
19 C. Chen, Y. Zhao, J. Wang, P. Zhu, Y. Tian, M. Xu, L. Wang 21 D. Tanaka, W. Kawakubo, E. Tsuda, Y. Mitsumoto,
and X. Huang, Micromachines, 2018, 9, 160.
20 A. B. Theberge, G. Whyte, M. Frenzel, L. M. Fidalgo,
R. C. R. Wootton and W. T. S. Huck, Chem. Commun.,
2009, 6225.
D. H. Yoon, T. Sekiguchi, T. Akitsu and S. Shoji, RSC Adv.,
2016, 6, 81862.
This journal is © The Royal Society of Chemistry 2020
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