# Identification and Isolation Techniques for Plant Growth Inhibitors in Rice
## 5 Isolation and Identification Techniques for PGIs in Rice
### Qualitative and Quantitative Analyses
#### 5.3.2 Chromatography  
Thin-layer chromatography (TLC) is a method for the rapid determination of the fingerprint compounds in the natural compound extraction based on the mobile phase composition and the water content of the silica stationary phase. To date, TLC has been developed to rotate planar chromatography (RPC), centrifugally accelerated thin-layer chromatography (CTLC), and overpressure layer chromatography (OPLC) to overcome the disadvantages of classical TLC. Normally, the thicknesses of the chromatography plates are 0.5-2 mm, with the maximum sample load for a 1.0 mm silica layer being about 5 mg.cm\({}^{-2}\). Optimization of mobile phases plays an important role in determining organic compounds in the mixtures, and n-hexane: ethyl acetate, n-hexane: acetone, and chloroform: methanol are frequently used as the mobile phases with various proportions [148]. The evidence of this technique in the isolation of allelbochemicals in rice is shown in Table 3.  
Pressure liquid chromatography techniques are the main form of liquid chromatography, occurring under 0.2 to 2.0 mPa. The principle of these techniques depends on the stationary-phase column, the mobile phases, and the specific samples. Other technical features which have been applied to identify natural compounds in rice are also briefly summarized in Table 3. High-pressure liquid chromatography (HPLC) is one of the main-stays in the isolation of the stage in natural products. In this technique, the particle size of the stationary phase is significant for separate processing, with its range being from 3 to 10 \(\upmu\)m, which is smaller compared to other liquid chromatography methods, such as low- or medium-pressure liquid chromatography. Regarding the particle size, the extracted plant compounds in the mobile phase can be pushed through the system by the high pressure. The interaction between the high surface area of the solutes and the high surface area of the solvent leads to improving the purifying ability of mixtures. The diameter of the column (from 10 to 100 mm), as well as the length of the column, depends on the compounds that need to be isolated [149; 150]. To date, a variety of pressure liquid chromatography techniques have been applied to determine the composition of natural compounds such as phenolic acids, flavonoids, and terpenoids owing to their structures, vapor temperature, and pressure [151]. In this section, the evidence also indicates that HPLC is the optimal method for obtaining simple phenols in rice and other plants by combining it with a spectroscopic section (Table 3) [152]. Hydrophilic interaction chromatography is the solution for the analysis of polar substances (HILIC). However, the separation mechanism of HILIC needs to be clear because HILIC separation has been obtained on adsorption and partition processes owing to the intermolecular force [150; 151]. Silica gel, silica-based polar bonded phases, particulate organic polymer materials, zwitterionic stationary phases, and monolithic HILIC stationary phases are the solutions used in the HILIC columns so far. Based on these mixtures, HILIC columns and the mobile phase will be chosen for suitable separation [153]. The applications of this technique in isolating allelbochemicals from rice are shown in Table 3. More specifically, the revolutionary separation technique is ultra-performance liquid chromatography (UPLC), which surmounted the limitations of previous liquid chromatography methods, with high resolution, speed, and sensitivity, due to its upgraded instruments and column technologies. Indeed, [PERSON] et al. [24] quantified MB by means of HPLC and ultra-performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS) in 2019. The limit of detection (LOD) and limit of quantification (LOQ) of MB were 0.48 ng/mL and 0.46 ng/mL, respectively, by using HPLC with a 250 mm \(\times\) 4.6 mm column, 10 \(\upmu\)m, and flow rate of 0.4 mL/min.