3. The procedure of affinity chromatography  

Usually, there are five steps during the affinity chromatography:

1) Sample injection
2) Adsorption of components of interest
3) Washing (Removal of impurities)
4) Elution of components
5) Regenerate the column

1) Sample injection  

    Before the injection of the components, we should pre-equilibrate the column with 3-5 volumes fresh solvent (buffer) through the column.     

    As shown in Figure 1, the component of interest is marked as the red ones. 

Fig.2 The first step of affinity chromatography: sample injection

 

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2) Adsorption of components of interest  

    Under a property slow flow rate, the interested component can be adsorbed to the ligands when they pass through the column. The other components, which have no affinity to the ligands, will be “pushed” to the end of the column by the solvent.

    If there are several components have affinity to the stationary phase, there will be a competitive adsorption on the ligands. The component, which has the strongest affinity to the ligands, will replace the components of weaker affinity to take the adsorption sites. Therefore, which have weaker affinity to the ligands, will also be “pushed” to the end of the column by the solvent.

Fig.3 The first step of affinity chromatography: adsorption of components of interest

 

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3) Washing (Removal of impurities)

    The usual treatment to remove the impurities is by washing the column with 3-5 volumes fresh solvent through the column.

Fig.4 The first step of affinity chromatography: washing

 

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4) Elution of components

   We often remove the adsorbed component of interest from the column by changing the previous solvent to other solvent, such as a higher salt concentration solution.

Fig. 5 The first step of affinity chromatography: elution of the components

 

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5) Regenerate the column

 The usual treatment to remove the impurities is by washing the column with 3-5 volumes fresh solvent through the column.

 

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