Yu Xie,Guoming Huang,Weiguo Hu,Yujun Wang,

1 The State Key Laboratory of Chemical Engineering,Department of Chemical Engineering,Tsinghua University,Beijing 100084,China

2 North China Pharmaceutical Group Co.,Ltd.,Shijiazhuang 052165,China

Keywords:Pendant drop Interfacial tension Microchannel Droplet size Piperacillin synthesis Mass transfer

ABSTRACT Piperacillin is a polar organic substance,and can reduce the interfacial tension of oil and water when dissolved in water.In this study,changes in dichloromethane–water interfacial tensions and microdroplet sizes during piperacillin synthesis from an aqueous solution of ampicillin and dichloromethane solution of 4-ethyl-2,3-dioxo-1-piperazine carbonyl chloride (EDPC) were observed using a pendent drop technique and a coaxial ring tube system with embedded high-speed camera,respectively.It was found that the rapid N-acylation reaction caused the piperacillin at the interface to synthesize rapidly and diffuse out slowly,resulting in the interfacial tension decreased from 19.5 mN.m-1 to 7.2 mN.m-1 rapidly and then increased slowly as the concentrations of ampicillin and EDPC were 0.05 mol.L-1 and 0.1 mol.L-1.Meanwhile,the increase in the concentration of EDPC increased the peak concentration of piperacillin at the interface,and the addition of ethyl acetate to the ampicillin solution promoted mass transfer and reduced the aggregation of piperacillin effectively.During synthesis,the interfacial tension decreased,leading to a change in droplet sizes in the micro-reaction system.The two-phase reaction was carried out in a coaxial ring tube,with ampicillin and EDPC solutions as continuous and dispersed phases,respectively.The reaction reduced the dripping flow area,and the addition of ethyl acetate to the ampicillin solution slightly affected the division of the flow pattern.Under the same flow conditions,the droplet sizes of the reaction group were smaller than those of the no reaction group.The experimental results demonstrated that the increase of the continuous phase,decrease in the dispersed phase flow rate,or increase in EDPC concentration making droplet sizes smaller,and the addition of ethyl acetate slightly affected droplet sizes.These findings are important for the design and optimization of piperacillin synthesis reactors.

1.Introduction

The synthesis of piperacillin (PRL) by ampicillin (AMP) and 4-ethyl-2,3-dioxo-1-piperazine carbonyl chloride (EDPC) through an N-acylation reaction[1]is the main route for industrial production of PRL,as shown in Fig.1.In a previous study[2],a three-feed membrane dispersion was designed and used to synthesize PRL with low impurity content,but the liquid–liquid microdispersion was not discussed in the reaction.In the system for PRL synthesis by the ampicillin chloride method,AMP in aqueous solution with pH approximately 8.7 is almost insoluble in dichloromethane (DCM),and EDPC in DCM is almost insoluble in water;therefore,the reaction occurs mainly at the DCM–water phase interface.The N-acylation reaction in PRL synthesis occurs rapidly,which causes a significant change in substrate concentration at the interface,and changes in the solution composition affect the liquid–liquid interfacial tensions [3–9].

H2O is a strong polar molecule,and DCM is a non-polar molecule.According to the principle of similar compatibility,the two are incompatible with each other and form a phase interface.Both PRL and AMP exist as carboxylates when dissolved in water under alkaline conditions.The effect of carboxylate on the interfacial tensions has been studied several times [10,11].PRL was obtained by introducing the more polar piperazinone acid into the amino group of AMP.The introduction of the polar group changes the antibacterial spectrum;thus,PRL can resist Pseudomonas aeruginosa[12,13].Meanwhile,changes in the molecular polarity lead to a change in the interfacial tension.

Fig.1.Main route for PRL synthesis.

The microdroplet size influences the mixing effect significantly.As the droplet size decreases,the specific surface area increases,and the mass transfer and reaction efficiency improve.In the micro-dispersion process,the influence of the interfacial tension is dominant for the droplet sizes.The change in interfacial tension will significantly impact the flow pattern distribution and dispersion scale of the liquid–liquid dispersion system.During the PRL synthesis,the reaction causes changes in the distribution of substances at the interface,which in turn affects the interfacial tensions of the liquid–liquid two-phase,resulting in changes in droplet sizes.In addition,the study of liquid–liquid microdispersion scale in the reaction is necessary for designing and optimizing high-efficiency microreactors.

The pendant drop equipment uses the shape characteristics of the pendant drop to calculate the liquid–liquid two-phase interfacial tension when the gravity is balanced with the interfacial tension,which can be used to measure the relationship between time and the interfacial tensions[14–18].Nowrouzi et al.[14]used the pendant drop method to study the effects of dissolved carbon dioxide and ions in water on the dynamic interfacial tension of water and oil in the process of carbonated smart water injection into oil reservoirs.Li et al.[19]observed the mass transfer characterization during biological catalytic hydration of acrylonitrile using a pendant drop equipment.The combination of a microchannel and high-speed microscopic camera system is a common method for studying micro-dispersion [20–22].Li et al.[23] used a T-junction and a high-speed CCD video camera to observe the droplet formation of a H2SO4/alkane system and discussed the effect of gravity.Xu et al.[24] proposed a microfluidic approach for rapid interfacial tension measurement by observing droplet sizes in coaxial ring tubes in different interfacial tension systems.

Herein,using a pendant drop equipment and a coaxial ring tube system with embedded high-speed camera,we studied the effects of solution concentration and addition of ethyl acetate on interfacial tension by pendant drop method,the effects of reaction and addition of ethyl acetate on the division of flow patterns in the coaxial ring tube,and the effects of reaction,solution concentration,flow rate and addition of ethyl acetate on droplet sizes in the coaxial ring tube.The work provided a novel basis for evaluating rapid reactions at the interface.

2.Materials and Methods

2.1.Materials and chemicals

AMP,PRL,EDPC (98%),ammonia water (NH3.H2O,25%),DCM(99.5%),and ethyl acetate(EA,99.5%)were purchased from Shanghai Aladdin Bio-Chem Technology Co.,Ltd.,China and used as received.Deionized water was used throughout the experiments.

2.2.Interfacial tension measurement via pendant drop method

Changes in interfacial tensions during PRL synthesis were measured using a pendent drop technique (OCAH200,DataPhysics Instruments GmbH,Germany).The pendant drop formation device is shown in Fig.2(a),which consisted of a syringe with a needle(outer diameter of 700 μm) and a transparent cuvette 20 mm × 20 mm × 20 mm.When a pendant droplet was formed,the microphotographic system immediately captured the morphology of the droplet.Using the basic principle of balance between interfacial tension and gravity,the backstage computer calculated and displayed the value of interfacial tension of the moment.The room temperature was maintained at 25 °C during the measurement.Subsequently,0.05 mol.L-1AMP aqueous solution (pH adjusted to～8.7 by NH3.H2O) was placed in the cuvette,and a DCM solution of different EDPC concentrations was put in the syringe.The needle was immersed in the AMP solution,and the EDPC solution was injected downward to form a pendant drop as large as possible.The interfacial tensions were measured and recorded.A picture of the pendant drop morphology is shown in Fig.2(b) as an example.

AMP aqueous solutions of 0.0025,0.005,0.01,0.025,0.05,and 0.1 mol.L-1were prepared,and the pH was adjusted to～8.7.The interfacial tensions of AMP/PRL aqueous solutions and DCM were measured according to the above method.Using 0.05 mol.L-1AMP aqueous solution as the solvent,different amounts of PRL were dissolved,and the pH was adjusted to～8.7 to obtain 0.05 mol.L-1AMP solutions with different PRL concentrations,marked as *PRL-0.05AMP solution.And then,the interfacial tensions of *PRL-0.05AMP solution were measured.

2.3.Sample component detection via high-performance liquid chromatography

High-performance liquid chromatography was used to detect the components of the AMP and PRL solutions.The liquid chromatography column was a Thermo ODS-15711 (5 μm,4.6 mm×250 mm)reverse-phase column.First,a 2 mg.ml-1of AMP solution and a 2 mg.ml-1of PRL solution were prepared,and the pH was adjusted to～8.7 by NH3.H2O.After the baseline of the liquid chromatograph stabilized,20 μl of the solution was introduced into the instrument and analyzed according to a preset procedure.

The liquid chromatography conditions were the same as those reported previously [2]:

Flow rate:1 ml.min-1;detection wavelength:220 nm;detection time:65 min;column temperature:25 °C;lotion:CH3OH:H2O=1:1;diluent:3.12 g L-1NaH2PO4:ACN=75:25.

Mobile phase A:H2O:3.12 g.L-1NaH2PO4:TBAOH=576:200:24,and then phosphoric acid was used to regulate the pH to 5.5.Finally,265 ml CAN was added and mixed well.Mobile phase B:H2O:3.12 g.L-1NaH2PO4:TBAOH=126:200:24,and then phosphoric acid was used to regulate the pH to 5.5.Finally,650 ml of CAN was added and mixed well.

Fig.2.(a) Schematic diagram of the pendant drop technique for measuring liquid–liquid interface tension and (b) pendant drop morphology during the measurement.

The mobile phase gradient table were as follows (Table 1):

Table 1Mobile phase gradient table

2.4.Online observation of droplet formation using a coaxial ring tube system with embedded high-speed camera

A schematic of the experimental device is shown in Fig.3,including three syringe pumps,three syringes,a microchannel,and an online observation system.Since the dispersed phase was not in contact with the wall surface during the dispersion process of the coaxial ring tube microchannel,the process was simple.Therefore,the coaxial ring tube microchannel was selected for further research.In the experiment,the two phases were controlled by the pumps,and an optical microscope (BX61,Olympus,Japan)equipped with a high-speed camera with a frequency of up to 2000 figures per second was used to observe and record the droplet sizes in the microchannel.

The main microchannel of the coaxial ring tube was provided by a glass capillary with an inner diameter of 1.2 mm,and a stainlesssteel needle(outer diameter of 220 μm;inner diameter of 160 μm)was embedded into the channel as the inlet for the dispersed phase.The AMP solution was injected into the main channel from the symmetrical microgrooves as the continuous phase,and the EDPC solution was injected from the stainless needle as the dispersed phase to form the liquid–liquid two-phase flow.The temperature was maintained at 25 °C during the experiment.

3.Results and Discussion

3.1.Interfacial tensions of examined solutions

Interfacial tensions of different concentrations of ampicillin and DCM,and different concentrations of piperacillin and DCM are shown in Fig.4.The interfacial tensions of different concentrations of AMP aqueous solution are shown as green dots,and the interfacial tensions of different concentrations of PRL solution are shown as black dots.It can be seen from the fitting curves that the inter-facial tension decreased with the increase of concentration.And the interfacial tension of PRL solution and DCM with the same concentration was smaller than that of AMP solution and DCM,indicating that PRL had a greater influence on the interfacial tension.This laid the foundation for the discussion of interfacial tension in the following.When PRL was synthesized during the reaction,the interfacial tension at the liquid–liquid interface changed significantly.

The interfacial tension has the following relationship with concentration [25]:

where γ,γ0represents the interfacial tension of solution and the interfacial tension when the concentration is 0,c represents the concentration of the solution,and A and B are constants related to the properties of the solution.Based on this,the interfacial tensions of the AMP solution and DCM were fitted as a blue line and could be calculated by the following equation:

where cAMPstands for the concentration of the AMP.The interfacial tension between water and DCM was 28.2 mN.m-1(25 °C).

The interfacial tensions of the PRL solution and DCM were fitted as a red line and could be calculated by Eq.(3):

where cPRLstands for the concentration of the PRL.

The addition of AMP or PRL to the water decreased interfacial tension because they had both hydrophilic and hydrophobic groups.After DCM pendant drop formation,they were adsorbed at the water-DCM interface,thereby reducing interfacial tension.AMP contained both hydrophilic groups,such as carboxyl group,and hydrophobic groups,such as benzene ring.It acted like a surfactant,like the carboxylate discussed in the foreword[10,11].PRL was a derivative obtained by introducing piperazinone acid with extreme polarity onto the amino group of AMP,which ability to break hydrogen bonds between water molecules at the interface was greater,so PRL could reduce interfacial tension more.At the same time,from the peak times of the chromatograms,the polarity of PRL was smaller.The 2.0 mg.ml-1AMP and PRL solution components were detected using high-performance liquid chromatography,and the obtained chromatograms are shown in Fig.5.Since reverse chromatography was used,components with strong polarities were the first to be flushed out of the column,while the components with weak polarities exhibited stronger retention on the column.As shown in the Fig.5,the characteristic peak of PRL in alkaline solution was about 8.7 min and that of AMP in alkaline solution was about 3.2 min.It was speculated that piperacillin had a greater affinity to oil phase at the water-DCM interface,which was also conducive to reducing interfacial tension.

Fig.3.Online observation device for liquid–liquid micro-dispersion using a coaxial ring tube system with embedded high-speed camera.

3.2.Changes in interfacial tensions during the reaction

Different concentrations of EDPC solution were injected into 0.05 mol.L-1AMP solution,and the changes in interfacial tensions were measured over time,as shown in Fig.6.The interfacial intension of the DCM and 0.05 mol.L-1AMP solution was 19.5 mN.m-1.As can be seen from the figure,the start time of the measurement was not the beginning of the reaction,because the reaction had already occurred during the formation of the pendant drop.After the pendant drop was formed,the interfacial tension decreased rapidly to the lowest point and then slowly increased to the highest point.When the EDPC solution was 0.1 mol.L-1,the minimum of the interfacial intension was 7.2 mN.m-1.As shown in Fig.6,the process could be divided into three stages:the stage of rapid decrease of interfacial tension,the stage of slow rise of interfacial tension,and the stage of stable interfacial tension.As discussed in Section 3.1,the increase of piperacillin at the interface led to the decrease of interfacial tension.And,the changes of interfacial tensions during the reaction corresponded to the process of the synthesis of piperacillin.The N-acylation reaction was a rapid reaction.Although there was no external agitation in the pendant drop process,the EDPC in the pendant drop was exhausted within 20 seconds,and the interfacial tension reached a minimum value.At this point,piperacillin at the interface was supersaturated and slowly diffused into the water,making the interfacial tension increase slowly until it reached equilibrium.When the concentration of EDPC pendant drops increased,more PRL was produced by the rapid N-acylation reaction at the interface,and the interfacial tension was reduced more by the aggregation of PRL.After the reaction was almost done,the diffusion of PRL was the main influence of interfacial tension,and the interfacial tension began to rise.It can be seen,PRL produced by the reaction of EDPC with high concentration significantly affected the PRL concentration in the cuvette and kept the stable interfacial tension at a low value.

Fig.4.Interfacial tensions of different solution and DCM.

3.3.PRL concentrations at the interface during the reaction

The interfacial tensions of the *PRL-0.05 mol.L-1AMP solution and DCM are shown in Fig.7.The graph shows that the increase in PRL concentration led to a decrease in interfacial tension.The corresponding relationship between *PRL-0.05 mol.L-1AMP and interfacial tensions can be obtained by data fitting:

where γ0=19.5 mN.m-1is the interfacial tension of the 0.05 mol.L-1AMP solutionand DCM.

During the reaction of the EDPC in the pendant drop with the AMP solution in the cuvette,the amount of PRL was much greater than that of EDPC;thus,the concentration and pH of the AMP solution were regarded as constant.According to Eq.(4),the apparent concentration changes of PRL at the interface can be calculated,as shown in Fig.8.From the concentration change curve of PRL,the reaction process of the pendant drop can be divided into three stages:the rapid reaction stage of AMP and EDPC,the slow diffusion stage of PRL,and the equilibrium stage.The pendant drop experiment results demonstrated that even when the droplet equivalent diameter was approximately 1 mm and the solution was in a static stage,the reaction could be completed quickly(within 20 s).In addition,the PRL concentration peak was reduced by EA.The concentration of the pendant drop surface changed quickly by adding EA,indicating that the addition of EA promoted the mass transfer during the process.When a pendant drop of DCM was formed,the EA in water phase would move across the oil–water interface and transfer to the oil phase,due to ethyl acetate was more easily dissolved in dichloromethane than in water.And the movement led to the external vibration in the liquid and thus promoted the mass transfer.The addition of EA effectively prevented the aggregation of the products,which was conducive to the efficiency of the reaction.

Fig.5.Chromatograms of 2.0 mg.ml-1 AMP solution (a) and PRL solution (b).

Fig.6.Interfacial tension variation graph during the reaction.

3.4.Division of flow patterns in coaxial ring tube

Because of the rapid N-acylation reaction at the liquid–liquid interface,a concentration gradient appeared at the interface,which caused changes in the physical properties at the interface,especially the interfacial tension.Interfacial tension is the main factor affecting flow pattern distribution of the liquid–liquid microdispersion.Using a high-speed microscope camera system,two different flow patterns were observed during the experiment of the coaxial ring tube:jetting flow and dripping flow,as shown in Fig.9.Dripping flow refers to the droplets formed by breaking near the inlet of the dispersed phase,which has extremely high monodispersity,and the generated droplets are relatively stable.Jetting flow refers to the droplets formed when the breaking position is at a certain distance from the inlet of the dispersed phase,which has poor dispersion.Herein,jetting flow was defined as the flow where the breaking position was at least three times the outer diameter of the needle from the inlet of the dispersed phase.

Fig.7.Interfacial tensions of *PRL-0.05 mol.L-1 AMP solution and DCM.

Fig.8.PRL concentrations with time during the reaction.

By comparing the changes of liquid–liquid micro-dispersion flow pattern in different systems(DCM–water;DCM–water containing 5%EA;0.1 mol.L-1EDPC solution–0.05 mol.L-1AMP solution;0.1 mol.L-1EDPC solution– 0.05 mol.L-1AMP solution containing 5% EA),the influence of reaction and mass transfer between phases on the division of flow patterns was discussed.In the coaxial ring tube,the flow pattern transition process was determined by the viscous drag force of the continuous phase,the inertial force of the dispersed phase,and the interfacial tension between the two phases.When the sum of the viscous drag force and the inertial force is greater than the interfacial tension,the flow pattern changes.In Fig.10,Udand Ucare the dispersed and continuous phase velocity,respectively.Fig.10(a) and (c) shows that the dripping area of the reaction group was greatly reduced compared to the non-reaction group,indicating that the existence of the reaction significantly reduced the interfacial tension of the liquid–liquid two phases.Comparing Fig.10(a) and (b),the addition of EA made the dripping area slightly larger because the interfacial tension of the two phases at the moment the droplets falling increased.The reaction group showed a similar phenomenon,as shown in Fig.10(c)and(d).During the reaction between the EDPC solution and the AMP solution in the coaxial ring tube,EA in the AMP solution passed through the interphase mass transfer into the dispersed phase.The existence of this interphase mass transfer caused a slight increase in the interfacial tension.

3.5.Effect of reaction on droplet sizes

Owing to the simple droplet formation method and uniform size of the dripping flow,the operating range in this work was controlled within the typical dripping flow area.The 0.05 mol.L-1AMP solution adjusted to pH～ 8.7 was used as the continuous phase,and a DCM solution of 0.1 mol.L-1EDPC was used as the dispersed phase.The continuous phase flow rate was fixed at 1000 μl.min-1,and droplet sizes are shown in Fig.11(a) as the dispersed phase flow rate changes.Fdand Fcare the dispersed and continuous phase flow,and ddrefers the droplet diameter.The dispersed phase rate was fixed at 50 μl.min-1,and droplet sizes are shown in Fig.11(b) as the continuous phase flow rate changes.Comparing the droplet sizes of the solvent group with that of the reaction group under the same flow rate condition,it was found that the droplet sizes of the reaction group were much smaller.The viscosity of the AMP solution was not much different from that of water because the AMP solution was dilute.Considering that the dispersed phase and the continuous phase flow rate were fixed,the viscous drag force of the continuous phase of the reaction group and the solvent group are almost the same.The occurrence of the reaction resulted in an increase in the PRL concentration and a decrease in the interfacial tension at the interface,which in turn led to a decrease in droplet size.

Fig.9.Two flow patterns in the coaxial ring tube observed by the high-speed microscope camera system:(a) dripping flow and (b) jetting flow.

Fig.10.Division of flow patterns in coaxial ring tube:(a)DCM–water;(b)DCM–water containing 5%EA;(c)0.1 mol.L-1 EDPC solution–0.05 mol.L-1 AMP solution;and(d)0.1 mol.L-1 EDPC solution– 0.05 mol.L-1 AMP solution containing 5% EA.

Fig.11.Effect of reaction on droplet sizes:(a) different flow rate of the dispersed phase and (b) different flow rate of the continuous phase.

3.6.Effect of reaction on apparent interfacial tensions at droplet shedding moment

As shown in Fig.11,for both solvent and reaction groups,the droplet size increased with an increase in the dispersed phase flow rate and decreased with an increase in the continuous phase flow rate.In the dripping flow area,the breakup dynamics are dominated by the force balance between the interfacial tension force and the viscous drag force [26,27].To better understand the effect of the reaction on the interfacial tension,a method of balancing the viscous drag force and the interfacial tension when the droplet shedding was used to calculate the apparent interfacial tension at the droplet shedding moment under the reaction conditions.At the droplet shedding moment,the interfacial tension Fσand the viscous drag force of the continuous phase FDwere calculated as follows:

where kDis a parameter related to the structure and material characteristics of the microchannel.Therefore,the liquid–liquid interfacial tension γrat the droplet shedding moment in the presence of the reaction can be calculated using the following equation:

Fig.12.Effect of the dispersed phase flow rate on apparent liquid–liquid interfacial tensions at droplet shedding moment under reaction conditions.

3.7.Effect of EDPC concentration in the dispersed phase on droplet sizes

A 0.05 mol.L-1AMP solution(pH 8.7) was used as the continuous phase,and different concentrations of EDPC solution were used as the dispersed phase in the coaxial ring tube.The droplet sizes were observed online using a coaxial ring tube system with embedded high-speed camera when they fell off the needle.For different EDPC concentrations,as the continuous phase flow rate increased,the viscous drag force increased and droplet size decreased.The results are shown in Fig.13(a),which is consistent with the conclusion of Section 3.5.Under the same flow rate conditions,the concentration of the EDPC solution increased,leading to a decrease in droplet sizes.As discussed in Section 3.6,PRL synthesis occurred rapidly.The amount of AMP in the continuous phase was excessive under experimental conditions;thus,as the concentration of EDPC solution increased,more PRL was formed at the interface.The flow rate was fixed;therefore,the mixing efficiency and PRL diffusion rate were almost the same.Therefore,when the EDPC concentration increased,the PRL synthesized at the interface increased,resulting in a decrease in the liquid–liquid interfacial tension,which decreased droplet size,as shown in Fig.13(b).

Fig.13.Effect of EDPC concentration in the dispersed phase on droplet sizes.

Fig.14.Effect of adding EA to AMP solution on droplet sizes.

3.8.Effect of adding EA to AMP solution on droplet sizes

The 0.05 mol.L-1AMP solution(pH～ 8.7),in which the solvent was a mixture of deionized water and EA at a volume ratio of 95:5,was used as the continuous phase,and a DCM solution of 0.1 mol.L-1EDPC was used as the dispersed phase.The droplet sizes observed in the reaction with and without EA at the same concentration were observed online.As shown in Fig.14,the reaction with EA exhibited slightly larger droplets than that without EA,indicating that the addition of EA slightly increased the liquid–liquid interfacial tension at the droplet shedding moment.This was consistent with the conclusion in Section 3.4.Sections 3.2 and 3.3 describe that the addition of EA increased mass transfer,which was beneficial to avoid the aggregation of products.This was beneficial to the reaction.In particular,considering the strict restriction of impurities in PRL production [2],the enhancement of micro-mixing might have a beneficial effect on reducing the occurrence of side reactions.

4.Conclusions

Using a pendent drop technique and a coaxial ring tube system with embedded high-speed camera,the interfacial tensions of liquid–liquid two-phase and droplet sizes during PRL synthesis by the ampicillin chloride method were determined.When a single pendent drop of 0.1 mol.L-1EDPC solution reacted with a large amount of 0.05 mol.L-1AMP solution,PRL was generated rapidly at the interface and diffused outward slowly,resulting in the interfacial tension of the liquid–liquid two-phase decreased rapidly from 19.5 mN.m-1to 7.2 mN.m-1and then increased slowly.When EA was added to the AMP solution,the micro-mixing was strengthened and the aggregation of the product was avoided effectively due to the introduction of the interphase mass transfer.When the synthesis was conducted in a coaxial ring tube,the reaction reduced the dripping flow area,and the addition of ethyl acetate to the ampicillin solution slightly affected the division of the flow pattern.Under the same flow conditions,the droplet sizes of the reaction group were smaller than those of the no reaction group.The increase in the continuous phase flow rate,decrease in the dispersed phase flow rate,or increase in the EDPC concentration would benefit to improve the formation of smaller droplets.Since the addition of EA slightly affected the sizes of the droplets and benefited mass transfer,the addition of EA in the reaction was considered advantageous.

Declaration of Competing Interest

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.

Acknowledgements

This work was financially supported by the National Key Research and Development Program of China (2019YFA0905100),the National Natural Science Foundation of China (21878169 and 21991102),and Tsinghua University Initiative Scientific Research Program (2018Z05JZY010).