Phase Transfer of AMIET-functionalized Gold Nanoparticles from Aqueous to Organic Solvents , the preparation of the desired particles in

: This paper presents a feasible and reliable phase transfer protocol for polyoxyethylene alkyl amine surfactant (AMIET)-coated gold nanoparticles (AuNPs) in aqueous media to chloroform using a pH-triggered method, through the liquid-liquid interface. In the initial stage, the colloidal aqueous dispersion is destabilized by pH adjustment towards the isoelectric pH of the nanoparticle, which promotes the separation of the particles from water. We further explored a mechanistic view of this phase transfer phenomenon, considering the orientation of hydrophilic and hydrophobic moieties depending on the nature of the surrounding solvent. It was proposed that the AMIET molecules bound to the AuNPs undergo conformational changes through phase transfer. Ultraviolet visible absorption spectra before and after the phase transfer reveal that the original morphology and dispersion states of the particles were preserved.

solvents requires more complicated and meticulous steps because of the low solubility of gold salts in the organic phase, 10 and the complexity in controlling the size and shape of AuNPs 11 .
In contrast, the synthesis of AuNPs in the aqueous phase is more prevalent because it is simple, rapid, and environmentally friendly 12 . Moreover, the synthesis in aqueous media has many well-defined preparation procedures 13 . Therefore, the synthesis of AuNPs in water is the most convenient and economical method, considering its applications. However, it is noteworthy to mention that the use of NPs in electrical elements requires the incorporation of metallic NPs that are free from contact with water, and are dispersed in an organic matrix as well 14,15 . The organic dispersion is also mandatory for the formation of a thin film monolayer of AuNP to weaken the interfacial energy between metal NPs 16,17 .
Furthermore, in organic catalysis, solution-processable optoelectronic applications, and composite materials, the nanoparticles are needed to be dispersed and stabilized in a nonaqueous liquid phase 18 . Therefore, the transfer of AuNPs, especially from aqueous to water-immiscible organic solvents, is often required to increase their range of application and also to take advantage of the preparation process in water. However, the colloidal stability of NPs remains the critical issue with their phase transfer 19 . It is often challenging to transfer NPs from a stable colloidal system to another phase. Although numerous promising approaches have been carried out for the successful phase transfer of NPs from the aqueous to the organic phase, one of the major drawbacks of these techniques is either exploiting post-synthesis ligand exchange 20 , adding of cosolvents 21 , or employing ionic-liquids 11,22 , which may disrupt the integrity of NPs in the second solvent. Several studies have been conducted using various methods for phase transfer. For instance, the centrifugation method was extensively studied by Lee et al. 23 , which is useful for organic solvents that are denser than water, and pH-sensitive zwitterionic amphiphiles were used for the recovery and redispersion of AuNPs 24, 25 . Imura et al. showed reversible phase transfer by changing the pH of the colloid 26 . Chakraborty and Christopher have also investigated the reusability of pH-responsive gold AuNP catalysts 27 . Despite their success, several critical issues exist, including incomplete transfer with few residual particles remaining in the aqueous phase and the instability of transferred NPs in non-aqueous solvents during storage. Additionally, most ligand exchange processes are applicable to very limited types of NPs 18,28 . Therefore, it is necessary to develop a more general and robust phase transfer strategy for the dispersion of the synthesized AuNPs in the organic phase.
In this study, AuNPs were produced via a single-step synthesis with the reduction of tetra chloroauric III acid by AMIET, a polyoxyethylene alkyl amine surfactant with a weak cationic character. In the industry, AMIET has been used for various applications as an antistatic agent, emulsifier, dispersant, detergent for textiles, and so on, but in this work, it acts as a reducing and capping agent for AuNP synthesis. Furthermore, we present an effective, straightforward phase transfer method for laboratory-synthesized AMIET-AuNPs without the addition of any specific chemical. The phase transfer of AuNPs was investigated by applying three strategies to establish a robust and repeatable phase transfer methodology for AMIET-AuNPs: simple vigorous shaking 3-4 times daily, centrifugation method, and pH-triggered phase transfer method. It was observed that the phase transfer of AMIET functionalized AuNPs depends on the particle s surface charge and its dispersion pH. Hence, this study attempted to destabilize the aqueous colloidal system by changing its pH to obtain nearly zero surface-charged particles 29 . Consequently, efficient phase transfer was promptly achieved at a state known as isoelectric pH. It has been suggested that the surface-bound ligand shell can undergo conformational changes depending on the surrounding solvents that favor the particles for phase transfer 30,31 . Ultraviolet-visible UV-vis spectroscopy and scanning transmission electron micrographs confirmed that the particles preserve their size and morphology while transferring to chloroform. Furthermore, the ligand arrangement in both phases or the orientational changes of surface-bound AMIET have been explained and well supported by their nuclear magnetic resonance NMR spectra.  Fig. 1 were obtained from Kao Corporation and used for the synthesis of AuNPs. Because AMIETs have been produced from natural sources for industrial purposes, each AMIET is essentially a mixture of polyoxyethylene alkyl amines with a certain number of x y in polyoxyethylene parts and distributed alkyl chain lengths. The safety data sheets indicate that AMIETs whose names began with 3 and 1 are polyoxyethylene hydrogenated tallow amines and polyoxyethylene cocoamines, respectively, and the last two digits express the total number of oxyethylene units i.e., for AMIET 320, x y 20 . Sodium hydroxide Kanto Chemical Co. Inc. , 37 hydrochloric acid Kanto Chemical Co. Inc. , and chloroform FUJIFILM Wako Pure Chemical Corporation were used as received. Ultrapure water obtained by Merck Direct-Q UV was used both as a solvent and for cleaning the glass wares throughout the experiments. Prior to use, the glass wares were immersed overnight in a 5 Cica Clean Kanto Chemicals solution and sonicated with ultrapure water twice for 15 min, followed by drying for 3 h at 100 in an electric oven.

Preparation of AuNPs
The AuNPs were prepared using a chemical reduction method. Initially, a fixed amount of AMIET and ultrapure water was heated in a three-necked round bottom flask Table 1 . When the temperature reached 80 , a desired amount of 48.6 mM hydrogen tetrachloroaurate III tetrahydrate aqueous solution yellow color was added to the flask. After a few minutes, the entire solution became wine red, indicating the reduction of gold chloride with the surfactant and the formation of AuNPs. At this stage, the mixture was allowed to reflux for 1 h to ensure the completion of the reaction while maintaining a temperature of 80 and a stirring rate of 100 rpm. Then heating was turned off and the mixture was allowed to cool to room temperature with continuous stirring. Finally, the synthesized AuNPs were stored in a clean glass container with a screw cap at room temperature.

Characterization
The UV-vis absorption spectra of the AuNPs were measured using a JASCO V-600 spectrometer with a 1 cm path length quartz cuvette within the range of 300 nm -700 nm, and baseline correction was performed with ultrapure water as reference. Zeta potential measurements were carried out by laser Doppler electrophoresis using a Zetasizer Nano ZS Malvern with a DTS1061-disposable cuvette. The pH of the AMIET-AuNP dispersion was adjusted using 0.01 M HCl or 0.01 M NaOH and measured using a pH meter Mettler Toledo . Transmission electron microscopy TEM observations were performed using a JEOL JEM 2010 at an accelerating voltage of 200 kV with a copper grid coated with an elastic carbon film ELS-C10 STEM Cu100P, OKENSHOJI . To prepare size distribution histograms, 100 particles were selected randomly from TEM images to estimate their diameter using ImageJ software. Proton nuclear magnetic resonance NMR spectra were recorded using an Agilent-NMR-vnmrs 500 spectrometer to understand the ligand arrangement on the particle surface upon phase transfer. In the preparation of NMR samples, the aqueous AMIET-AuNPs were centrifuged and D 2 O was added to the sedimented part, which resulted in sufficient shimming to take the NMR spectrum. The AMIET-AuNPs dispersed in CDCl 3 were prepared as follows. Ultrapure water was added to the concentrated AMIET-AuNPs dispersed in chloroform. The mixture was vigorously shaken and allowed to settle for approximately 30 min, after which a complete separation between the water and chloroform phases was observed, and a layer of AMIET was also observed with the naked eye at the interface. Then, the water and AMIET layer were removed using a micropipette. As long as the layer was noticed, washing was repeated continuously. The obtained AMIET-AuNPs were dried overnight, and CDCl 3 was added to prepare the NMR samples.

Formation of AMIET-coated AuNPs
Research has been carried out previously on the strong ability of tertiary amines to form AuNPs 32 . Similar to this research, AMIET as a tertiary amine contributed to the formation of AMIET-coated AuNPs in the present work, as confirmed by the appearance of a red color and surface plasmon resonance absorption in the UV-vis absorption spectra of the reaction solutions, as well as TEM observation of the formed particles. A photograph of the AMIET320-AuNP aqueous dispersion is shown in Fig. 3 a , as an example. All the reaction solutions showed a typical red color, as shown in the photograph after the formation of AuNPs. The typical wavelength of the absorption peak due to surface plasmon resonance for spherical gold nanoparticles varies from 520 to 530 nm 33 . Therefore, the peaks observed in the UV-vis spectra of the as-prepared AMIET-AuNPs Fig. 2 Table 2 were assigned to the characteristic surface plasmon resonance absorption of spherical AuNPs. The complete conversion of Au 3 to Au 0 was confirmed by the absence of a peak near 300 nm, which is the characteristic peak of Au 3 due to ligand metal charge transfer 34 , although the corresponding wavelength region of the spectra is not shown in Fig. 2 a . A TEM image of the as-prepared AMIET320-AuNPs is shown in Fig. 4. TEM observation also confirmed the formation of spherical AuNPs of a few to 20 nm diameter for all AMIET-AuNPs. Figure 5 shows the size distribution histograms obtained through TEM image analysis of AMIET-AuNPs dried from aqueous dispersions. Although all AMIET-AuNPs were polydisperse and AMIET302-AuNPs were smaller than those of the other AMIET-AuNPs, no difference was observed in the phase transfer using the pH-triggered method.

Determination of isoelectric point of AMIET-AuNPs
The factors affecting the zeta potential include pH, ionic strength, and concentration of the nanocolloid. However, pH is the most leading parameter in zeta potential measurements of NPs in an aqueous medium 29 . The plots of zeta potentials as a function of pH are shown in Fig. 6 for different AMIET-AuNPs. The derived isoelectric points from the plots are 5.4 for AMIET320-AuNP, 7.9 for AMIET302-, 105A-, and 105-AuNPs, and 9.0 for AMIET 102-AuNP. Because AMIETs are mixtures of polyoxyethylene alkyl amines as mentioned above, the isoelectric point would be determined as averaged or apparent one due to of the complicated effects of the ingredients; thus, it is not easy to fully understand the difference in isoelectric point on the basis of molecular structure. However, the number of ethyleneoxy EO units seems to have an effect on the isoelectric point because AuNPs prepared using AMIET320 with 20 EO units showed a much lower isoelectric point Fig. 3 Photographs of AMIET320-AuNP dispersions: a as-prepared AMIET-AuNPs in water, b at isoelectric pH, followed by phase transfer to c chloroform.

Phase transfer to organic solvent
Initially, the phase transfer experiments were performed by a simple addition method in which 5 mL of chloroform was added to 5 mL of AuNP dispersion, shaken vigorously, and then left for a few days with shaking 3-4 times daily , in order to observe the phase transfer process. The phase transfer occurred by this method, but it was also observed that the holding periods for phase transfer were not identical. In addition, more than 90 of the particles moved towards the chloroform phase after 7 days, according to the decrease in intensity of the plasmon peak in the UV-vis spectra, but achieving complete transfer was expected to take a long time. The second attempt was made by the centrifugation method, where 5 mL of chloroform was added to 5 mL of AuNP dispersion and the mixture was centrifuged twice for 80 min using 10,000 rpm rotation. This method efficiently reduced the time for the phase transfer as compared to the simple addition and shaking method, but not all the cases recorded a complete transfer. An alternative approach is the pH-triggered method, in which the pH of the AuNP dispersion is adjusted to the isoelectric point. Using this method, it was confirmed that the AMIET rapidly functionalized AuNPs and efficiently transferred to the organic phase. The UV-vis spectra of AMIET-AuNPs in chloroform after phase transfer at the isoelectric points are shown in Fig. 2 b . At the isoelectric point, the AuNPs may be in an unstable state, depending on the nature of the colloids Fig. 3 b , but the phase transfer easily occurred immediately after the addition of the organic solvent Fig. 3 c . The complete transfer of AuNPs was confirmed by UV-vis analysis of the aqueous phase after the transfer process, where no plasmonic absorption was observed data not shown . In chloroform, as the refractive index of the solvents increases n water 1.33, n chloroform 1.45 , a small red shift of 3 nm is typically observed 35 . However, not all the peak positions of the spectra in Fig. 2 b , which are summarized in Table 2, follow the expected change due to solvent exchange, suggesting that the dispersion state of AMIET-AuNPs would change de-  pending on the solvent. Nevertheless, except for AMIET 302-AuNPs, no significant change in the plasmon peak position was observed, which indicates that the dispersion state of AuNPs did not change significantly between water and chloroform. Even for AMIET302-AuNPs, although the peak position was shifted to a relatively large extent, the particles were stably dispersed in chloroform for a long time.

Interpretation of 1 H-NMR results
The orientation of the hydrophobic and hydrophilic moieties of AMIET on AuNPs in both phases was evaluated by 1 H-NMR spectra, based on the relative signal intensity depending on molecular mobility 30,36 . Figure 7 presents the 1 H-NMR spectra of AMIET and AMIET-AuNPs in CDCl 3 and D 2 O. The signals of the terminal methyl head peaks α and methylene group peaks β of long alkyl chains were observed with high intensity whether AMIET molecules were free or bound to AuNPs in CDCl 3 , suggesting that the chains were exposed in the solvent. The signals of EO chains peaks γ also appeared in AMIET dissolved in CDCl 3 , which is due to the fact that the EO unit is not extremely hydrophilic because it contains two methylene units 37 and thus soluble in organic solvents. However, the signals γ were weakened for the AMIET320-AuNP dispersion in CDCl 3 and almost disappeared for the other AMIET-AuNPs in CDCl 3 , implying tight packing of the EO parts on the AuNPs. The weak appearance of the signal γ in the spectrum for the AMIET320-AuNP dispersion in CDCl 3 arises from some parts of the longer EO chains i.e., x y 20 which are not tightly fixed on the surface of the AuNPs. These observations indicate that the alkyl moiety is oriented toward CDCl 3 , whereas the EO chains are oriented away from CDCl 3 Fig. 8 a , which makes the dispersion feasible and ensures nanoparticle stability in chloroform. In contrast, for AMIET molecules in D 2 O, the signals α and β were much weaker, and the signals γ appeared relatively strong. This tendency was enhanced for AMIET-AuNPs in D 2 O, except in the case of AMIET102-AuNP. The exception might be due to the fewer number of EO groups along with their close attachment to the AuNP surface. According to the observed changes in the relative intensity, the most  Phase Transfer of AMIET-functionalized Gold Nanoparticles plausible orientation of AMIET molecules in D 2 O would be the reversed one from the case in CDCl 3 Fig. 8 b .

Conclusion
In this work, the preparation of AuNPs using AMIET via a simple heating method in an aqueous phase, and the phase transfer of the synthesized AuNPs to a chloroform phase without the addition of a surface modifier, were demonstrated. It was found that the phase transfer efficiently progressed while preserving the shape and size of AuNPs by the pH-triggered method, where the pH of the aqueous phase was adjusted to an isoelectric one. The changes in 1 H-NMR spectra imply that the phase transfer would accompany the orientational switching of hydrophobic and hydrophilic groups of AMIET molecules depending on the environmental solvent. The present work not only confirms the emerging role of AMIET ligands but also predicts the very promising direction of using AMIET in direct synthesis as well as the phase transfer of AuNPs. Moreover, the pH-triggered phase transfer method would be advantageous for other combinations of aqueous/organic solvents other than water/chloroform, regardless of the density of the organic solvent. Experiments are underway on the phase transfer of AuNPs to nonaqueous solvents other than chloroform.