**2.** *In situ* **microwave irradiation NMR spectrometer**

The *in situ* microwave NMR spectrometer employed consisted of a solid-state NMR spectrometer and a microwave generator that was capable of transmitting 1.3 kW continuous wave (CW) and pulsed microwaves at a frequency of 2.45 GHz (**Figure 1A**). This spectrometer enables NMR signals to be obtained without the interference of microwaves, while radio waves and microwave irradiation are

#### **Figure 1.**

*Microwave Heating - Electromagnetic Fields Causing Thermal and Non-Thermal Effects*

with Co particles under microwave irradiation [24].

proteins in the native and denatured states [31].

determined using the relation between 1

that of the bulk solution [34].

high-resolution observation of a 1

To characterize the microwave heating mechanism with the molecular resolution, microwave-irradiation nuclear magnetic resonance (NMR) spectroscopy was first developed by Naito et al. [25]. The characteristic that microwave heating causes a rapid temperature jump was used to obtain state-correlated two-dimensional (2D) NMR spectra between liquid crystalline and isotropic phases. This enabled

H state-correlated 2D NMR spectra of the liquid state toward the liquid crystalline state. The local dipolar interaction of individual protons in the liquid crystalline state can be examined via high-resolution resonance in the isotropic phase [26–30]. The resulting data can also be used to obtain state-correlated 2D NMR spectra of

*In situ* microwave irradiation NMR spectroscopy was developed later [32], and the microwave heating process was observed in liquid crystalline samples [32, 33] and ethanol-hexane mixed solution [34]. The *in situ* temperature of the bulk solution was

perature is defined as chemical shift calibrated temperature (CSC-temperature) and is measured for individual protons. In N-(4-methoxybenzylidene)-4-butylaniline (MBBA) molecules, H-C=N (7′), and CH3-O (α') protons showed significantly higher CSC-temperatures than the bulk temperature in the isotropic state [33]. In the ethanol-hexane mixed solution, OH proton showed lower CSC-temperature than

Molecular dynamics (MD) simulations have recently been conducted to investigate the atomic-scale properties of molecular systems under an electric field. Tanaka and Sato investigated the heating process of water and ice under microwave irradiation. They observed that the rotational motion of the water was delayed due to the microwave electric field, and the energy is transferred to the kinetic and

H dipolar pattern in the cross section of the

H chemical shift and temperature. This tem-

H-1

and collision, which increases entropy and the energy is dissipated through the system. From a thermodynamic point of view, this microwave energy converts to heat energy (thermal effect) and work energy such as volume change (non-thermal effect). However, the detailed molecular mechanisms associated with thermal and non-thermal microwave effects on the chemical reaction rates have not yet been fully elucidated. In particular, the non-thermal microwave effect has not yet been well characterized [7]. In the field of chemistry, microwave heating is widely used to accelerate organic synthesis reactions [2–4, 7–14], reduce polymerization reaction times [15–18], and enhance the activity of enzymes in the field of biological chemistry [19–21]. The majority of accelerated reactions achieved in this manner can be mainly explained by the thermal microwave effect [7, 22]. Non-thermal microwave effects have also been identified and the thermal and non-thermal microwave effects can be distinguished [23]. The non-thermal microwave effects have recently been demonstrated by the observation of an increasing polymerization reaction rate under microwave electric fields and a decrease in the rates under microwave magnetic fields [18]. Nevertheless, non-thermal microwave effects at the molecular level are still controversial [7]. In particular, non-thermal microwave effects have been considered as a direct interaction of the electric field with polar molecules in the reaction medium which is not explained with a macroscopic temperature effect [2, 22]. The presence of an electric field leads to effects on the orientation of dipolar molecules or intermediates, and thus changes the pre-exponential factor A or the activation energy in the Arrhenius equation for certain types of reactions [2, 22]. As a specific microwave heating effect, non-equilibrium localized heating is defined as the generation of isolated regions with a much higher temperature than the bulk solution. This has been observed in liquid–solid systems under microwave irradiation, such as in the case of dimethyl sulfoxide (DMSO) molecules in contact

**170**

1

*(A) In situ microwave irradiation solid-state NMR spectrometer which consists of a superconducting magnet, an NMR spectrometer (CMX infinity 400, Chemagnetics), and a microwave transmitter (IDX, 1.3 kW, Tokyo electron Co., ltd.). CW and pulsed microwave are gated by the pulse programmer of the NMR spectrometer. Microwaves are generated and transmitted through a waveguide and coaxial cable, and finally introduced to the microwave resonance circuit in the probe. (B) Equalizing microwave resonance circuit consisting of an inductor and capacitor. The sample tube was located inside the capacitor. (C) Schematic diagrams of microwave resonance and radio wave resonance circuits. The top view indicates the names of components and the side view shows the dimensions of the components. Adapted with permission from [32]. Copyright (2015) Elsevier Inc.*

simultaneously applied. Therefore, *in situ* NMR observation is possible under microwave irradiation using this *in situ* microwave irradiation NMR spectrometer. A 3 mm wide flat copper ribbon was used to form the capacitor of the microwave resonance circuit (**Figure 1B**) and was wound coaxially inside the radio wave circuit to increase isolation during microwave irradiation, which reduced interference between microwaves and radio waves (**Figure 1C**) [32]. The microwave resonance circuit was tuned to 2.45 GHz and the radio wave circuit was set to 398 MHz for <sup>1</sup> H NMR using a sweep generator. Microwaves were generated from the microwave generator and transmitted through a waveguide and a coaxial cable, and finally, the microwaves were guided to the resonator circuit at the probe head. <sup>1</sup> H NMR signals were obtained using 5.0 μs of 90° pulse under microwave irradiation with the *in situ* microwave irradiation NMR spectrometer. The temperature of the sample was varied using the temperature control unit of the spectrometer. Samples were packed in an inner glass tube to insulate them from thermal contact with the outer glass tube. During microwave irradiation, the temperature was controlled at 0°C for the ethanol-hexane mixed solution and at 50°C for MBBA in the isotropic state.
