# Identification and Isolation Techniques for Plant Growth Inhibitors in Rice
## 5 Isolation and Identification Techniques for PGIs in Rice
### Extraction, Separation, Isolation, and Purification Techniques  
#### 5.2.1 Extraction and Separation Techniques  
Extraction is the primary and most important stage to isolate, identify, and quantify phytochemicals from the rice. The raw materials have diverse and complex ingredients that are obtained with different solvents of increasing polarity in order to exhaustively extract as many active compounds in rice as possible [122; 123]. To improve the conventional extraction techniques, modern extractions such as pressurized liquid extraction (PLE), microwave-assisted extraction (MAE), solid-phase microextraction (SPME), and superheated liquid extraction were applied to extract and isolate organic compounds in plants. This section focuses on reviewing PLE, MAE, and SPME techniques. Pressurized liquid extraction (PLE) is also known as accelerated solvent extraction (ASE). This methodology is applied for solid extraction, using common solvents at elevated temperatures (temperature range from 50 to 200 \({}^{\circ}\)C) and pressures (under the range from 10 to 15 MPa). Extract processing is performed under pressure in order to maintain the solvent in a liquid phase. The PLE overcomes other methods due to pressure flexibility. The analyte is pushed out from the cell with the solvent by the pressure, and the cell pores are filled out quickly by the pressure flow, giving higher efficiency in extraction. The high temperature increases solubility, enhances diffusion, and increases the mass transfer of the analyte, shortening the extraction process by some minutes. Compared with traditional extraction methods, PLE has some advantages, such as lower solvent consumption and higher extraction efficiency. Until now, no evidence has revealed the degradation of temperature-sensitive compounds due to the high temperature [124; 125]. On the other hand, PLE and supercritical fluid extraction (SFE) have also been applied to extract the same samples, and the results were similar [126]. However, the obtained extracts need to be cleaned up because the PLE has to use solvents for the extraction. The PLE was used to extract allelochemicals in rice, which are shown clearly in Table 2.  
Microwave-assisted extraction (MAE) with microwave energy (ranging from 300 to 300,000 MHz) has been applied for extraction through accelerating the heating speed by direct heat for the extraction of solvents. Two microwave energy extraction technologies have been developed, either using closed systems under controlled temperature and pressure (PMAE: pressurized microwave-assisted extraction) or open systems under controlled atmospheric pressure (FMAE: focused microwave-assisted extraction). The aim of the application of these technologies is to extract compounds selectively and rapidly. Thereby, this technique also remarkably decreases organic solvent consumption. However, the FMAE is suitable in the case of volatile compounds, although the reaction time is longer compared to the PMAE due to the reducing temperature of the systems. The principle of PMAE and PLE is the same, namely using a difference in energy to heat the solvents [127; 128]. MAE was used to extract allelochemicals in rice, which are shown clearly in Table 2.  
Solid-phase extraction (SPE) is the process of purifying natural product extracts using adsorption compounds in the solid phase. Natural product purification has high efficiency if the SPE is integrated into automated sample preparation and isolation systems. Silica gel, ion-exchanged resins, mixed-mode materials, or reversed-phase materials are used to pack SPE cartridges. The unwanted compounds are retained in the solid phase and the wanted compounds are eluted. The required compounds retained in the solid phase will be eluted by other compatible solvents [128]. Indeed, the purification of samples by using SPE before liquid or gas chromatography analysis makes the determination of the structure and configuration of compounds more efficient [129; 130].