# 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
#### 5.2.2 Isolation and Purification Techniques  
Thin-layer chromatography (TLC) is a principal chromatographic technique that is widely applied in screening natural products and supporting the isolation and purification of natural compounds [135]. Its advantages are its simplicity and time- and cost-effective [135]. In order to improve the TLC technique, high-performance thin-layer chromatography (HPTLC) was developed, which is simple, rapid, and enhances the separation of compounds [135]. The detection sensitivity of HPTLC is 5 to 10 times higher compared to classical TLC [135]. Additionally, this improved TLC technique is efficient in the quantitative analysis of compounds [135]. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Rice Samples** & **Compounds** & **Techniques** & **Conditions** & **Tested Plants** & **Ref** \\ \hline \multirow{4}{*}{Rice plants} & \multirow{4}{*}{(S)-Limonene} & \multirow{4}{*}{SPME} & Solid phase (SPME): & \multirow{4}{*}{\begin{tabular}{} \end{tabular} } \\  & & & & divinylbenzene & Bacterial blight \\  & & & (DVB)/carbox & (_Xanthomonas oryzae_ pv. & [20] \\  & & & (CAR)/polydimethylsiloxane & _Oryza_) & [20] \\ \hline \multirow{4}{*}{Rice husk} & \multirow{4}{*}{\begin{tabular}{} \end{tabular} } & \multirow{4}{*}{\begin{tabular}{} \end{tabular} } & HexarLeOAc; & \multirow{4}{*}{\begin{tabular}{} \end{tabular} } \\  & & & CHCl\({}_{3}\)-MeOH & & \\  & & & & _crus-gall_), tall & \\  & & & & in a \(5\times 60\) cm column & goldenrod (\(S\). & [99] \\  & & & & followed by 200–400 mesh & _altisima_), and Lettuce & \\  & & & & in a \(2\times 50\) cm column & (_Lactuca sativa_) & \\ \hline \multirow{4}{*}{Rice grains} & \multirow{4}{*}{Phenolics} & \multirow{4}{*}{UAE} & MeOH:H\({}_{2}\)O (80:20, \(v/v\)) & \multirow{4}{*}{\begin{tabular}{} \end{tabular} } \\  & & & At 45 \({}^{\circ}\)C, in 25 min, cycle & \multirow{4}{*}{\begin{tabular}{} \end{tabular} } \\  & & & 0.4 s\({}^{-1}\), ultrasound & \\  & & & amplitude 47\% & \\ \hline \multirow{4}{*}{Rice grains} & \multirow{4}{*}{Phenolics} & \multirow{4}{*}{PLE} & EtOAc:MeOH (60:40, \(v/v\)) & \multirow{4}{*}{\
\begin{tabular}{} \end{tabular} } \\  & & & Pressurized 200 atm and & \\  & & & then heated 190 \({}^{\circ}\)C in 10 & \\  & & & min with three cycles & \\ \hline \multirow{4}{*}{Rice grains} & \multirow{4}{*}{Phenolics} & \multirow{4}{*}{MAE} & 100\% MeOH & \multirow{4}{*}{
\begin{tabular}{} \end{tabular} } \\  & & & Solve to sample (10:1) & \\ \cline{1-1}  & & & At 185 \({}^{\circ}\)C, 20 min, cycle & \\ \cline{1-1}  & & & 0.4 s\({}^{-1}\), microwave power & \\ \cline{1-1}  & & & 1000 W & \\ \hline \hline \end{tabular}  
* SPME: solid-phase microextraction; UAE: ultrasound-assisted extraction; MAE: microwave-assisted extraction; PLE: pressure-liquid extraction; EtOAc: ethyl acetate; CHCl\({}_{3}\): chloroform; MeOH: methanol.  
\end{table} 
Table 2: Extraction techniques used in the separation of PGIs in rice. Column chromatography (CC) is a well-known method for separating and purifying compounds from plant extracts [136]. The column contains a stationary phase with silica gel [136]. Through the column, the mobile phase separates the compounds based on their affinities from the stationary and mobile phases [136]. After that, the isolated components can be collected along with the mobile phase [136]. CC can be listed with two common types, including normal CC and flash CC [136]. Reverse phase chromatography (RPC) is based on a partitioning mechanism to induce separation effects [137]. Separation in RPC is determined through the reversible adsorption and desorption of solute molecules with different levels of hydrophobicity to the hydrophobic stationary phase [137]. Most reverse-phase separations are performed in five steps consisting of starting conditions, sample adsorption, the initiation of desorption, the termination of desorption, and generation [137]. This technique was originally developed in the 1960s to separate small organic molecules.