**2.2. Protein A or protein G affinity chromatography**

as altering pH of the targets and ligands that interact by weakly acidic or basic groups. Changing pH can lead to the alteration in the conformation of the target or ligand. Either increasing or decreasing of pH value can be used for this purpose but decreasing of pH is commonly preferred. Irreversibly denaturation of target, ligand or support may occur in this step. Collection of the eluted target in a neutral pH buffer and regeneration the column as soon as possible after the elution step can overcome this problem [8]. Changes in ionic strength induced by high salt solutions are a second way for nonspecific elution. Disruption of ionic bonds can be achieved by this method but hydrophobic interactions are promoted. Chaotropic salts (NaCl, MgCl2 or LiCl) are useful for altering retention of targets. They distrupt the stability of water and interfere with hydrophobic interactions [8,23]. The main advantage of using either chaotropic salt of a change in ionic strength is that this usually leads to gentle elution of the target in an active form [8]. Denaturating agents such as urea, guanidine hydrochloride and sodium dodecyl sulfate which dissociate hydrogen bonds can also be used for elution. Sodium dodecyl sulphate (SDS) contains both hydrophobic and strong ionogenic groups and binding to hydrophobic regions results in a layering of negative charges on the protein's surface, causing irreversible unfolding of the structure. The denaturating effect of these solutions limits their usage. They should be only used in analytical applications if the ligand is quite stable or in preparative applications if both the ligand and target are relatively stable and it is enable to recover their activity after such elution [8,23]. Organic solvents in the mobile phase are also used in some cases such as using of 1-propanol in chiral affinity separations in order to improve solute retention and produce narrow peaks for good resolution. Polyols like ethylene glycol are also utilized in affinity separations [8]. In order to select the elution buffer several ap‐ proaches can be followed. However the best way is that the buffer should be selected based on information in the literature, structure of the ligand, target and past experiences with these

The type of ligand can be used to divide affinity techniques into various subcategories such

Immunoaffinity chromatography is one of the most popular techniques of affinity derivatived method and it enables to produce ligands in case the ligand required is not available [7]. In this technique, stationary phase comprises of an antibody or antibody-related agent [1]. It is possible to isolate variable subtances using this technique due to high specifity of antibodies [1]. It is reported that immunoaffinity chromatography may be used for natural food contam‐

On the purpose of purification using antibodies as ligands, antibodies initially are immobilized on a support. In order to,bind the ligand on the surface of the support properly, protein A and G are usually used as a bridge which provides enough space for the ligand-protein binding.

as lectin, immunoaffinity, dye ligand etc. These techniques are placed as below [1].

substances [8].

88 Column Chromatography

**2. Types of affinity chromatography**

inants such as aflatoxins, fumonisins and ochratoxins [11].

**2.1. Immunoaffinity chromatography**

Protein A is produced by *Staphylococcus aureus* while protein G is of group G *Streptococci*. These ligands are capable of binding to many types of immunoglobulins at around neutral pH and they dissociate in a buffer with a lower pH [1]. Protein A binds to the immunoglobulin G (IgG) obtained by human and other mammalian species with high specificity and affinity. In some cases protein G may be used instead of protein A [24]. These two ligands differ in their ability to bind to antibodies from different species and classes. Strong specifity and binding properties to immunoglobulins of protein A and protein G serve them as good ligands for the seperation of immunoglobulins. Protein A and protein G have use as secondary ligands for the adsorption of antibosies onto the support material in immunoaffinity applications. This method may also be employed in case high antibody activity or replacement of the antibodies in the affinity chromatography is needed [1].
