**3.5 Crystallization method**

Crystallization is the process of solute precipitation from mother liquor with complex components, and it is an effective method to prepare pure substances. The initial crystallization is often impure and needs to crystallize again, which is called recrystallization. It is a method to separate compounds from the mixture by using the difference of solubility of each component in the solvent. Crystallization is one of the important technologies for plant chemists to prepare pure compounds.

When the content of a phytochemical is very high in one plant, crystals can be obtained by cooling or slightly concentrating the extract after extraction with appropriate solvent. For example, see [16].

Selecting suitable crystallization solvent is the key of the crystallization method. The ideal solvents for crystallization should possess the following characteristics: high solubility for the components to be purified at high temperature, low solubility at low temperature, insoluble for the impurities at high and low temperature, or soluble for the impurities at high and low temperature, moderate boiling point, no chemical reaction with the components to be crystallized, safe, low price, easy to obtain, and so on. Solvents commonly used for crystallization are methanol, ethanol, acetone, ethyl acetate, acetic acid, pyridine, etc. When crystals cannot be obtained with a single solvent, the crystallization operation can be carried out with a mixture of two or more solvents. Mixed solvents generally consist of two miscible

solvents, one of which has high solubility for the component to be crystallized, and the other has low solubility. Firstly, the sample to be crystallized is heated and dissolved in as few solvents as possible with high solubility. Then the second solvent with low solubility is added to the hot solution to make it turbid. Then the first solvent is added to dissolve the sample. The solution reaches saturation at this point and crystallizes when it is cooled. The purity of crystallization can be preliminarily identified by the crystal form, color, melting point, melting range, thin layer chromatography, paper chromatography, etc.
