*3.6.2 Gel chromatography (exclusion chromatography, molecular sieve chromatography)*

Molecular sieve is the main principle of gel chromatography, which can separate mixture compounds according to the pore size of the gel and the molecular size of the compounds. Gel is a kind of solid material with a porous network structure. The molecules of the separated substances are different in size, so their ability to enter the gel is different. When the mixture solution passes through the gel column, the molecules smaller than the gel pores can enter the gel interior freely, while the molecules with larger size than the gel pores cannot enter the gel, and only pass through the gel particle gaps. Therefore, different movement rates are emerged. The molecules with large sizes are not excluded, and the retention time is shorter. The molecules with small sizes are detained because of its diffusion into the pores, thus the retention time is longer. There are many kinds of commercial gels, dextran gel and hydroxypropyl dextran gel are used most commonly [18].

#### *3.6.3 Ion exchange chromatography*

It is to separate chemical constituents according to the difference of dissociation degrees. In this method, ion exchange resin is applied as stationary phase and water or solvent mixed with water as mobile phase. The ionic components existing in the

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

*3.6.4 Macroporous adsorption resin chromatography*

washed out with distilled water before using.

*3.6.5 Partition chromatography*

polar molecular compounds.

**3.7 New technologies and methods**

*3.7.1 High performance liquid chromatography (HPLC)*

mobile phase are absorbed by ion exchange resin after ion exchange reaction. Ion exchange chromatography is suitable for the separation of ionic compounds, such as alkaloids, amino acids, organic acids, peptides, and flavonoids. The ability of ion exchange reaction between compounds and ion exchange resins mainly depends on the compounds' dissociation degree and the amount of electric charges. If the dissociation degree of a compound is high (acidic or alkaline), it is easily exchanged on resins and difficult to elute. Therefore, when the compounds with different degree of dissociation are exchanged on the resin, the compounds with lower degree of dissociation are eluted before those with higher degree of dissociation [19].

It is a chromatographic method which combines the principle of adsorption and molecular sieve. Its chromatographic behavior possesses reversed-phase properties. Macroporous resin is a kind of solid macromolecule material with no dissociable group and porous structure and is insoluble in water. It is widely used in the separation and enrichment of natural compounds because of its stable physical and

In practical work, the water solution of the mixture to be separated is usually washed by water, water-containing alcohol solution with low to high concentration. The mixture can be separated into several components. The regeneration of macroporous adsorbent resin is convenient. It is often washed by 1 mol/L hydrochloric acid and 1 mol/L sodium hydroxide solution, respectively, first, then washed by distilled water to neutral, and stored in methanol or ethanol. The alcohol should be

It is a kind of chromatography method to separate components by using different partition coefficients between stationary phase and mobile phase, which are immiscible liquids. Partition chromatography could be divided into normal phase chromatography and reverse phase chromatography. The polarity of stationary phase is stronger than that of mobile phase in normal phase partition chromatography, which is mainly used to separate polar and moderately polar molecular compounds. Carriers commonly used in normal phase distribution chromatography include silica gel, diatomite, cellulose powder, etc. Silica gel with water content of more than 17% can be used as a carrier for partition chromatography because of its loss of adsorption. It is the most widely used carrier for partition chromatography. In reverse phase partition chromatography, the polarity of mobile phase is stronger than that of stationary phase. The commonly used stationary phase is octadecylsilylated silica (ODS). The mobile phase is usually methanol-water or acetonitrilewater system, which is mainly used for the separation of nonpolar and moderately

High performance liquid chromatography (HPLC) is a rapid separation and analysis technology developed on the basis of conventional column chromatography. Its separation principle is the same as regular column chromatography, including adsorption chromatography, gel chromatography, partition chromatography, ion exchange chromatography, and other methods. HPLC columns are produced

chemical properties (insoluble in acids, bases, and organic solvents).

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

*Phytochemicals in Human Health*

chromatography, paper chromatography, etc.

flavonoids, anthraquinones, tannins, etc. [17].

**3.6 Classical chromatographic methods**

*3.6.1 Adsorption chromatography*

*chromatography)*

*3.6.3 Ion exchange chromatography*

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 is the most commonly used method for the separation of chemical constituents of natural products. It possesses advantages of high separation efficiency, rapidity, and simplicity. By choosing different separation principles, different operation modes, different chromatographic packings, or applying various chromatographic methods jointly, the separation and purification of various types of phytochemicals

It is a kind of chromatography based on the difference of adsorptive capacity of adsorbents to different compounds. The commonly used adsorbents include silica gel, alumina, activated carbon, polyamide, and so on. Silica gel adsorption chromatography is widely used, and it is suitable to the separation of most of the plant chemical constituents. Alumina adsorption chromatography is mainly used for the separation of alkaline or neutral lipophilic components, such as alkaloids, steroids, and terpenoids. Activated carbon is mainly used for the separation of water-soluble substances, such as amino acids, carbohydrates and some kinds of glycosides. Polyamide, which allows the separation to take place based on the formation of kinds of hydrogen bonds, is mainly used for the separation of phenols, quinones,

Molecular sieve is the main principle of gel chromatography, which can separate mixture compounds according to the pore size of the gel and the molecular size of the compounds. Gel is a kind of solid material with a porous network structure. The molecules of the separated substances are different in size, so their ability to enter the gel is different. When the mixture solution passes through the gel column, the molecules smaller than the gel pores can enter the gel interior freely, while the molecules with larger size than the gel pores cannot enter the gel, and only pass through the gel particle gaps. Therefore, different movement rates are emerged. The molecules with large sizes are not excluded, and the retention time is shorter. The molecules with small sizes are detained because of its diffusion into the pores, thus the retention time is longer. There are many kinds of commercial gels, dextran gel

It is to separate chemical constituents according to the difference of dissociation degrees. In this method, ion exchange resin is applied as stationary phase and water or solvent mixed with water as mobile phase. The ionic components existing in the

could be achieved. It can also be used for the identification of compounds.

*3.6.2 Gel chromatography (exclusion chromatography, molecular sieve* 

and hydroxypropyl dextran gel are used most commonly [18].

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mobile phase are absorbed by ion exchange resin after ion exchange reaction. Ion exchange chromatography is suitable for the separation of ionic compounds, such as alkaloids, amino acids, organic acids, peptides, and flavonoids. The ability of ion exchange reaction between compounds and ion exchange resins mainly depends on the compounds' dissociation degree and the amount of electric charges. If the dissociation degree of a compound is high (acidic or alkaline), it is easily exchanged on resins and difficult to elute. Therefore, when the compounds with different degree of dissociation are exchanged on the resin, the compounds with lower degree of dissociation are eluted before those with higher degree of dissociation [19].
