**3. Isolation and purification of phytochemicals**

The separation of phytochemicals is a process of isolating the constituents of plant extracts or effective parts one by one and purifying them into monomer compounds by physical and chemical methods. Classical isolation methods, including solvent extraction, precipitation, crystallization, fractional distillation, saltingout, and dialysis, are still used commonly at present. On the other hand, modern separation technologies such as column chromatography, high performance liquid chromatography, ultrafiltration, and high performance liquid drop countercurrent chromatography also play an important role in the separation of phytochemicals. This section describes the common methods and their specific applications in isolation of phytochemicals.

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*Analytical Methods of Isolation and Identification DOI: http://dx.doi.org/10.5772/intechopen.88122*

It is carried out according to the different acidity and alkalinity of each component in the mixture. Water-insoluble alkaline organic components, such as alkaloids, could react with inorganic acids and form salts, which can be separated from nonalkaline and water-insoluble components. Acid components with carboxyl or phenolic hydroxyl groups can be salted by bases and dissolved in water. Components with lactone or lactam substructures can be saponified and dissolved in water and then isolated from other water-insoluble components. The total extract can be dissolved in lipophilic organic solvents (ethyl acetate is commonly used) and extracted respectively with acid water and alkali water, and then the total extract would be divided into acidic, alkaline, and neutral parts. Of course, the total extract can also be dissolved in water and extracted with organic solvents after adjusting the pH value. The alkalinity or acidity of the fractions are different and can be separated further by pH gradient extraction. When using the acid and basic solvent method, attention should be paid to the strength of acidity or alkalinity, the contact time with the separated components, heating temperature, and time, so as to avoid the structural changes of some compounds under severe conditions or the chemical structures cannot be restored

This method is to achieve the separation aim based on the different polarity of each component in plant extracts and the different partition coefficients in two-phase solvents. Generally, different two-phase solvent systems are selected according to the polarity of components in plant extracts. For example, the components with strong polarity can be separated by n-butanol-water system, the components with medium polarity can be separated by ethyl acetate-water system, and the components with weak polarity can be separated by chloroform (or ether)-water system. During the operation, the plant extract should be dissolved by water firstly, and then the solution or suspension is extracted in a separating funnel with different organic solvent which is not miscible with water based on the polarity difference. Usually, the extract was extracted with petroleum ether (or cyclohexane) firstly, then ethyl acetate (or chloroform), and finally with water saturated n-butanol, as shown in **Figure 1**. Petroleum ether layer contains lipid-soluble compounds with low polarity. Ethyl acetate layer contains medium polar compounds such as monoglycosides, flavonoids, and compounds with more polar functional groups. N-butanol layer contains compounds with strong polarity, such as oligoglycosides and other water-soluble components. Compounds in water layer possess strongest polarity, such as glycosides with more glycosyl groups, carbohydrates, amino acids, proteins, and

It is a method based on the formation of precipitation of some phytochemicals by reaction with specific reagents, or the precipitation of some components from the solution by adding specific reagents, which can reduce the solubility of some components in the solution. The precipitation reaction must be reversible if the target components are required to form precipitation. While if the components are nontarget, the precipitation generated will be removed, so the precipitation reaction can be irreversible. According the addition of reagents or solvents, this method

*3.1.1 Acid and basic solvent method*

**3.1 Solvent method**

to the original states.

*3.1.2 Polarity gradient extraction method*

other water-soluble compounds.

could be classified as follows [11].

**3.2 Precipitation method**
