# Identification and Isolation Techniques for Plant Growth Inhibitors in Rice
## 5 Isolation and Identification Techniques for PGIs in Rice
### Qualitative and Quantitative Analyses  
#### 5.3.1 Spectroscopic Methods  
UV-visible (UV-vis) spectroscopy can be applied for the identification and quantification of specific groups of compounds in a pure form or in a mixture. Notably, aromatic compounds are strong chromophores in a wide range of UV wavelengths. Natural compounds including rice PGIs (e.g., phenolics, flavonoids, anthocyanins, tannins, etc.) have been detected by UV-vis spectroscopy [138], among which, phenolics, flavonoids, and anthocyanins can be determined with absorbances of 280, 320, and 520 nm, respectively. In addition, this technique requires a short time and low cost [139].  
Infrared spectroscopy (IR) is based on the vibrational changes inside a substance under IR radiation exposure. Different kinds of bonds (e.g., C-C, C-O, O-H, N-H, etc.) in a molecule have various vibrational frequencies. These bonds can be determined by detecting the characteristic frequency absorption band in the IR spectrum [139]. Fourier transform-infrared spectroscopy (FT-IR) is an improved IR technique with a high-resolution analytical tool to determine the chemical components and clarify the compound structure [139]. In another report, near-infrared spectroscopy (NIRs) equipped with UV-vis could evaluate the degradation of thermal rice oil [140]. A review by [PERSON] et al. showed that FT-IR has been used to elucidate the thermal deterioration mechanism of bio-oil of rice hus owing to it providing information about functional groups rapidly and accurately [141]. The models of NIRs have been developed to predict protein, amylose, crude oil, and fiber content in rice [119].  
In mass spectrometry (MS), compounds can be bombarded with electrons or lasers, thereby converting them to charged ions. A mass spectrum is recorded as the relative abundance of fragmented ions against the ratio of mass/charge of these ions. Accordingly, the relative molecular mass (molecular weight) can be calculated. In addition, elucidation of the compound structure can be conducted by applying tandem mass spectrometry (MS/MS). Therefore, LC equipped with MS systems has been improved to support compound identification more rapidly and accurately in extracted samples without the use of standard substances [142; 143; 144; 145]. Recently, LC-MS has been one of the most frequently used methods to identify and quantify some allelochemicals, such as phenolics and phenyl amides (Table 3).  
Nuclear magnetic resonance (NMR) spectroscopy is based on the magnetic characteristics of atomic nuclei. By reporting differences among various magnetic nuclei, NMR can determine the positions of these nuclei in a molecule. In particular, atoms and their amounts present in neighboring groups can be indicated [138]. Based on that, the structures of isolated and purified substances applying TLC, LC, and CC can be subsequently confirmed by NMR [138]. In 2019, the structures of momilactone A, B, E, and 7-ketostigmasterol in the crude extracted rice huselectrospray ionization (ESI), high-resolution mass spectrometry (HR-MS), and infrared spectroscopy (IR) [99].  
X-ray crystallography is the most powerful technique that can identify the atomic and three-dimensional (3D) structure of target compounds from small crystals to large molecules [146]. X-ray crystallography is the only method to figure out the absolute configuration of chiral molecules, and results from crystallographic studies [146]. In this study, there are four major steps, including crystallization, data collection, structure solution, and refinement/validation [146]. The X-ray crystallography method directly indicates information on the compound structure at the atomic level with high accuracy [147].