*3.2.2 Exclusive reagent precipitation method*

Some reagents could react selectively with certain chemical constituents to produce reversible precipitation, and the separation aims are achieved, which is called the exclusive reagent precipitation method. For example, alkaloid precipitation reagents such as Reynolds ammonium salt can precipitate after reacting with alkaloids, which can be used to separate alkaloids and nonalkaloids, or watersoluble alkaloids and other alkaloids. As another example, reactions of cholesterol and sterol saponins could form precipitation, which can separate them from triterpene saponins. Additionally, gelatin can precipitate tannins, which can be used to separate or remove tannins. In practical application, appropriate precipitation reagents should be selected according to the properties of target constituents and impurities in plants.

### *3.2.3 Salting out method*

Adding inorganic salts to a certain concentration or saturated state in the water extract of plants can reduce the solubility of some components in water, thus they could be separated from water-soluble compounds. The inorganic salts commonly used for salting out are sodium chloride, sodium sulfate, magnesium sulfate, ferric

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

**3.3 Dialysis method**

**3.4 Fractional distillation method**

**3.5 Crystallization method**

appropriate solvent. For example, see [16].

sulfate, etc. For example, extractions of tetrandrine from *Daemonorops margaritae* and berberine from *Berberis poiretii* could be achieved by salting out with sodium chloride or ammonium sulfate. Some water-soluble substances, such as protoanemone, ephedrine, and matrine, are often extracted with organic solvents after

It is a method to let substances selectively penetrate through natural or synthetic semi-permeable membranes (or dialysis bags) under the action of concentration difference, pressure difference, or potential difference, so as to achieve the purpose of separation, classification, purification, or concentration. For example, when saponins, proteins, polypeptides, polysaccharides, and other substances in plants are separated and purified, dialysis can be used to remove inorganic salts, monosaccharides, and other impurities. On the contrary, large molecular impurities can also be left in the semipermeable membrane, while small molecular substances can be separated and purified through the semi-permeable membrane into the solution outside the membrane [14].

Fractional distillation is a method of separating components in liquid mixtures based on their different boiling points. It is usually categorized into atmospheric, vacuum, molecular distillation, and so on. It is mainly used for the separation of volatile oils and some liquid alkaloids in plants. For example, the boiling points of the two alkaloids in total alkaloids of *Cicuta virosa,* coniine, and conhydrine are 166–167°C for the former and 226°C for the latter, which are quite different from each other, and then they can be separated by the fractional distillation method. Generally, if the boiling point difference of compounds in liquid mixtures is above 100°C, the separation can be achieved by repeated distillation of the solution. If the boiling point difference of compounds is below 25°C, the fractionation column is needed. The smaller the boiling point difference is, the finer the fractionation device is needed [15].

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

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

adding a certain amount of salt to the water extract. For example, see [13].

sulfate, etc. For example, extractions of tetrandrine from *Daemonorops margaritae* and berberine from *Berberis poiretii* could be achieved by salting out with sodium chloride or ammonium sulfate. Some water-soluble substances, such as protoanemone, ephedrine, and matrine, are often extracted with organic solvents after adding a certain amount of salt to the water extract. For example, see [13].
