**2.4 Thin-layer chromatography (TLC)**

It is the most convenient way to eradicate individual constituents from a mixture of compounds by using stationary phase and mobile phase, and the formation of a single spot actually indicates the presence of pure compound [34].

The spotted TLC paper was placed into the TLC chamber in such a way that the spot can stay just above the surface of the solvent system. After running of the solvent through a specified limit, then the operation was discontinued, and the TLC paper is dried in air. By treating with specified vanillin-sulfuric acid spray reagent, the paper was observed under UV light of different wavelengths to locate spots, so that can easily explore different types of medicinal constituents [35]. The paper was inserted into an iodine chamber to confirm about the spot is formed, and at last, I identified only single spot for the obtained crystal, which was examined under UV light and iodine chamber (**Figure 5**).
