*1.3.2. Covalent immobilization methods*

Covalent immobilization is the most popular method in affinity chromatography. In this method, it is necessary to activate the ligand and/or the support first. Activation of the ligand can be conducted when it is desired to couple this ligand through a specific region. An example is the creation of aldehydes in the carbohydrate regions of an antibody for its attachment to a support that contains amines or hydrazide groups. The use of an activated support is more common for ligand immobilization but tends to be less specific in nature. Examples include the immobilization of proteins through their amine groups to supports activated with *N*hydroxysuccinimide or carbonyldiimidazole. The support used for covalent immobilization must meet several requirements. First, sufficient number of groups for activation and ligand attachment should be. Hydroxyl groups on the support are employed in most covalent coupling methods. Depending on how its surface is activated, a support can be used to immobilize ligands through their amine, sulfhydryl, hydroxyl, or carbonyl groups, among others.


primary amines, but the cyanate ester is more reactive than the imidocarbonate. The CNBr method utilizes relatively mild conditions for ligand attachment, making it suitable for many sensitive biomolecules. But one problem with this approach is that the isourea linkages obtained by the reaction of CNBr with the support are positively charged at a neutral pH. This means that these groups can act as anion exchangers and nonspecific binding can be occur. Other problems with this method include the toxicity of CNBr, requiring the use of adequate safety precautions during the activation process, and the leakage of ligands that can result from CNBr-activated supports (Figure 12).

**Figure 12.** Cyanogen bromide immobilization method patway [8]

*1.3.1. Noncovalent immobilization technique*

technique can be subdivided as follow;

G for the adsorption of antibodies.

*1.3.2. Covalent immobilization methods*

others.

between a metal ion and electron donor groups.

immobilization.

78 Column Chromatography

The simple adsorption of ligand to surface, binding to a secondary ligand, or ligand immobi‐ lization through a coordination complex can be classified as this type of immobilization. This

**a.** Nonspecific Adsorption; It is based on the attachment of ligand to support that has not been specifically functionalized for covalent attachment. Adsorption of the ligand to a support depends on the chemical characteristics of both the ligand and support. Columbic interactions, hydrogen bonding, and hydrophobic interactions involve in this type of

**b.** Biospecific Adsorption; In this type of noncovalent immobilization method the ligand of interest bind to a secondary ligand attached to the support. Although a variety of secondary ligands can be used for this purpose, two of the most common are avidin and streptavidin for the adsorption of biotin-containing compounds and protein A or protein

**c.** Coordination Complexes; A coordination complex can be used to prepare an immobilized ligand in some cases. This is used to place metal ions into columns for immobilized metalion affinity chromatography (IMAC) which is based on the formation of a complex

Covalent immobilization is the most popular method in affinity chromatography. In this method, it is necessary to activate the ligand and/or the support first. Activation of the ligand can be conducted when it is desired to couple this ligand through a specific region. An example is the creation of aldehydes in the carbohydrate regions of an antibody for its attachment to a support that contains amines or hydrazide groups. The use of an activated support is more common for ligand immobilization but tends to be less specific in nature. Examples include the immobilization of proteins through their amine groups to supports activated with *N*hydroxysuccinimide or carbonyldiimidazole. The support used for covalent immobilization must meet several requirements. First, sufficient number of groups for activation and ligand attachment should be. Hydroxyl groups on the support are employed in most covalent coupling methods. Depending on how its surface is activated, a support can be used to immobilize ligands through their amine, sulfhydryl, hydroxyl, or carbonyl groups, among

**1.** Amine-Reactive Methods; Amine groups is often used for the immobilization of proteins and peptides. Specific methods are cyanogen bromide method, reductive amination, *N-*

**a.** Cyanogen Bromide Method ; The cyanogen bromide (CNBr) method was the first technique used on a large scale for immobilizing amine-containing ligands and involves the derivatization of hydroxyl groups on the surface of a support to form an active cyanate ester or an imidocarbonate group. Both of these active groups can couple ligands through

hydroxysuccinimide technique, and carbonyldiimidazole method.

**b.** Reductive amination (also known as the Schiff base method); Reductive amination couples ligands with amine groups to activated periodate is used to oxidize diol groups on the support's surface to give aldehydes. This can be performed directly on carbohy‐ drate-based supports like dextran or cellulose. However, materials like silica or glass must first be treated to place diols on their surface. This can be accomplished by reacting the silica or glass with γ-glycidoxypropyltrimethoxysilane, followed by acid hydrolysis. When an amine-containing ligand reacts with the aldehyde groups, the resulting product is known as a Schiff base. Since this is a reversible reaction, the Schiff base must be converted into a more stable form. This is achieved by including sodium cyanoborohy‐ dride in the reaction mixture. Cyanoborohydride is a weak reducing agent that converts the Schiff base into a secondary amine without affecting the aldehydes on the support. After the coupling reaction is completed, the remaining aldehyde groups can be removed by treating the support for a short period of time with a stronger reducing agent (i.e., sodium borohydride) or by reacting these groups with an excess of a small aminecontaining agent (e.g., ethanolamine). The Schiff base method is relatively easy to perform and often gives a higher ligand activity than other amine-based coupling methods. This also results in ligands that have stable linkages to the support and that can be used for long periods of time. However, there are some disadvantages of this method. One is the need to work with relatively hazardous agents such as sodium cyanoborohydride and sodium borohydride. Thus, care must be taken to perform this technique with proper ventilation and safety precautions. The use of sodium borohydride for the removal of excess aldehyde groups must also be carried out with caution, since the use of conditions that are too harsh may result in the loss of ligand activity.


while others can be used with a variety of materials. The final selection among these approaches will often depend on the type of ligand being immobilized, the support desired for this ligand, and the conditions that can be tolerated by both the ligand and

NH2 + <sup>N</sup> <sup>O</sup> <sup>C</sup> (CH2)n <sup>C</sup> <sup>O</sup> <sup>N</sup>

O

NH - Ligand

O

O

C (CH2)n C O N

O O

DMF

NHS activated support

pH 7-8 Ligand - NH2

NH C (CH2)n C O O O

O O

O O

Affinity Chromatography and Importance in Drug Discovery

O

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81

**2.** Sulfhydryl-Reactive Methods; The use of sulfhydryl groups on ligands is another approach for preparing affinity supports. If a ligand has a free sulfhydryl group on its surface, using this group is advantageous, since it often provides site-specific immobili‐ zation and a cleavable product. If the ligand is a protein or peptide that has no free sulfhydryl groups but that does have a disulfide bond, this bond can be reduced to allow ligand attachment. It is also possible to introduce sulfhydryl groups on a ligand by thiolating aminesor carboxyl groups. A support can be activated in several ways for the immobilization of ligands through sulfhydryl groups. Unlike amine-reactive methods,

support during the immobilization process.

**Figure 13.** *N*-Hydroxysuccinimide immobilization method [8]

amine-containing

NH

matrix

**e.** Other methods; One example is the use of cyanuric chloride (or 2,4,6-trichlorotriazine) to activate hydroxyl- or amine-containing supports for ligand attachment. Cyanuric chloride has been widely employed as a cross-linking agent and as a reagent for protein modification. It has three reactive acyl-like chlorines, each of which has a different chemical reactivity. The first chlorine is reactive toward hydroxyls and amines at 4°C and pH 9. After the first chlorine reacts, the second requires a slightly higher temperature for its reaction (20°C), and the third chlorine needs an even higher temperature (80°C). Other techniques for amine-containing ligands include the azalactone, divinylsulfone, bisoxy‐ lane, ethyldimethylaminopropyl carbodiimide, and tresyl chloride-tosyl chloride meth‐ ods. Some of these methods are specific for certain supports (e.g., the azalactone method), Affinity Chromatography and Importance in Drug Discovery http://dx.doi.org/10.5772/55781 81

**Figure 13.** *N*-Hydroxysuccinimide immobilization method [8]

and often gives a higher ligand activity than other amine-based coupling methods. This also results in ligands that have stable linkages to the support and that can be used for long periods of time. However, there are some disadvantages of this method. One is the need to work with relatively hazardous agents such as sodium cyanoborohydride and sodium borohydride. Thus, care must be taken to perform this technique with proper ventilation and safety precautions. The use of sodium borohydride for the removal of excess aldehyde groups must also be carried out with caution, since the use of conditions

**c.** *N*-Hydroxysuccinimide Method; The N-hydroxysuccinimide (NHS) method is another technique often employed when immobilizing biomolecules through amine groups. This gives rise to the formation of a stable amide bond. There are a number of ways a support can be activated with NHS. The relative ease with which activated supports can be prepared is one advantage of the NHS method. But the fast hydrolysis of NHS esters tends to compete with the immobilization of ligands. This rate of hydrolysis increases with pH and is particularly important when dealing with dilute protein solutions. The half-life of these NHS groups at pH 7 and 0°C is approximately 4 to 5 h and decreases to as little as

**d.** Carbonyldiimidazole Method; Carbonyldiimidazole (CDI) can also be used to activate supports for the immobilization of amine-containing ligands. This reagent can react with materials that contain hydroxyl groups to produce an acylimidazole, which forms an amide linkage as the result of the interaction with primary amines on a ligand. Supports with hydroxyl groups will react with CDI to produce an imidazolylcarbamate. The reaction of imidazolylcarbamata with primary amines proceeds at pH 8.5 to 10.0. The CDI method is relatively simple and easy to perform. In addition, supports that have been activated by production imidazolylcarbamate groups are more stable to hydrolysis than those activated by the NHS method. A CDI-activated support is stable when stored in dry dioxane, with a half-life of greater than 14 weeks. Another advantage of this method is that the amide linkages formed by this technique (as well as those created by the NHS method) are more stable than the isourea linkages obtained by CNBr immobilization. One disadvantage of the CDI method is that it tends to produce ligands with a lower activity

**e.** Other methods; One example is the use of cyanuric chloride (or 2,4,6-trichlorotriazine) to activate hydroxyl- or amine-containing supports for ligand attachment. Cyanuric chloride has been widely employed as a cross-linking agent and as a reagent for protein modification. It has three reactive acyl-like chlorines, each of which has a different chemical reactivity. The first chlorine is reactive toward hydroxyls and amines at 4°C and pH 9. After the first chlorine reacts, the second requires a slightly higher temperature for its reaction (20°C), and the third chlorine needs an even higher temperature (80°C). Other techniques for amine-containing ligands include the azalactone, divinylsulfone, bisoxy‐ lane, ethyldimethylaminopropyl carbodiimide, and tresyl chloride-tosyl chloride meth‐ ods. Some of these methods are specific for certain supports (e.g., the azalactone method),

than alternative techniques (e.g., reductive amination) (Figure 14).

that are too harsh may result in the loss of ligand activity.

10 min at pH 8.6 and 4°C (Figure 13).

80 Column Chromatography

while others can be used with a variety of materials. The final selection among these approaches will often depend on the type of ligand being immobilized, the support desired for this ligand, and the conditions that can be tolerated by both the ligand and support during the immobilization process.

**2.** Sulfhydryl-Reactive Methods; The use of sulfhydryl groups on ligands is another approach for preparing affinity supports. If a ligand has a free sulfhydryl group on its surface, using this group is advantageous, since it often provides site-specific immobili‐ zation and a cleavable product. If the ligand is a protein or peptide that has no free sulfhydryl groups but that does have a disulfide bond, this bond can be reduced to allow ligand attachment. It is also possible to introduce sulfhydryl groups on a ligand by thiolating aminesor carboxyl groups. A support can be activated in several ways for the immobilization of ligands through sulfhydryl groups. Unlike amine-reactive methods,

to form a reactive ester, which can react with primary amine groups on the support. The second part of this process involves combination of the haloacetyl-activated support with a ligand containing a sulfhydryl group. This reaction proceeds by nucleophilic substitu‐ tion and produces a thioether. The resulting bond is comparable to an amide linkage in stability. Although the reactivity of haloacetyl-activated supports toward sulfhydryls is relatively selective, these can react with methionine, histidine, or tyrosine under appro‐ priate conditions. If the immobilization is carried out above pH 8, amines can also react

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**b.** Maleimide Method; Maleimides are another group of reagents employed for the selective coupling of a ligand through sulfhydryl groups. These tend to be more selective than a haloacetyl for such a reaction. The activation of a support with a maleimide is accom‐ plished by using a homobifunctional or heterobifunctional cross-linking agent. One agent employed for this purpose is bis-maleimidohexane (BMH), which is a homobifunctional cross-linker with a maleimide group on both ends. The first of these groups can react with a support that has a sulfhydryl group. After the excess BMH has been washed away, the

**c.** Pyridyl Disulfide Method ; Pyridyl disulfide (or 2,2′-dipyridyldisulfide) is a homobifunc‐ tional cross-linking agent used for immobilizing ligands with sulfhydryl groups to supports that contain sulfhydryls on their surface. Activation of the support is accom‐ plished by disulfide exchange between the sulfhydryl groups on the support and pyridyl

**d.** Other Methods; Divinylsulfone (DVS) can be used to activate a hydroxylcontaining support by introducing a reactive vinylsulfonyl group on its surface at pH 10 to11. This support can then be reacted with ligands that contain sulfhydryl, amine, or hydroxyl groups, with the rate of this reaction following the order –SH > –NH > –OH. Although the resulting bond for a sulfhydryl group is labile, the linkage for amine-containing ligands

**3.** Hydroxyl-Reactive Methods; A number of methods have been used to couple ligands through hydroxyl groups. However, unlike many amine- and sulfhydryl-reactive methods, techniques for hydroxyl-containing ligands are not that selective. For example, the divinylsulfone method can be used for coupling an amine-, sulfhydryl-, or hydroxylcontaining ligand. Many supports used in affinity chromatography already contain hydroxyl groups on their surface. One way for the activation of these groups is to introduce bisoxirane (epoxy) groups. The most frequently used oxirane for this purpose is 1,4-butanediol diglycidyl ether, which contains two epoxy groups. One of the epoxy groups can react with the hydroxyl groups on a support while the other is used for coupling ligands containing sulfhydryl, amine, the reactivity of the terminal epoxide to other groups follows the order –SH > –NH > –OH. Strong alkaline conditions (pH 11) allow coupling by this method through hydroxyl groups, while amines and sulfhydryl groups can react at a lower pH (pH 7 to 8). Cyanuric chloride is another agent used for attaching a hydroxyl-containing ligand to a support. In this method, this can only be used effectively in the absence of amine groups due to the higher reactivity of these groups. As before‐

maleimide at the other end can react with a sulfhydryl group on a ligand.

disulfide, giving rise to the release of pyridyl-2-thione.

with these supports.

is more stable.

**Figure 14.** The carbonyldiimidazole immobilization method [8]

where hydroxyl groups on the support are generally used, most sulfhydryl-reactive methods require the introduction of an amine,carboxyl group, or some other intermediate site onto the support. For example, silica cannot be used directly with sulfhydryl-reactive methods but must be reacted with aminopropyltriethoxysilane or mercaptopropyltrime‐ thoxysilane to convert it into a suitable form.There are various approaches that can be used to immobilize ligands with sulfhydryl groups. The following subsections examine some of these techniques, including the haloacetyl, maleimide, and pyridyl disulfide methods.

**a.** Haloacetyl Method; The haloacetyl method uses supports that contain iodoacetyl or bromoacetyl groups for the immobilization of ligands through sulfhydryl residues. These supports are usually prepared via the reaction of an amine-containing material with iodoacetic or bromoacetic acid in the presence of ethyldimethylaminopropyl carbodii‐ mide (EDC) at pH 4 to 5. EDC reacts with the carboxylic acid in iodo- or bromoacetic acid to form a reactive ester, which can react with primary amine groups on the support. The second part of this process involves combination of the haloacetyl-activated support with a ligand containing a sulfhydryl group. This reaction proceeds by nucleophilic substitu‐ tion and produces a thioether. The resulting bond is comparable to an amide linkage in stability. Although the reactivity of haloacetyl-activated supports toward sulfhydryls is relatively selective, these can react with methionine, histidine, or tyrosine under appro‐ priate conditions. If the immobilization is carried out above pH 8, amines can also react with these supports.


where hydroxyl groups on the support are generally used, most sulfhydryl-reactive methods require the introduction of an amine,carboxyl group, or some other intermediate site onto the support. For example, silica cannot be used directly with sulfhydryl-reactive methods but must be reacted with aminopropyltriethoxysilane or mercaptopropyltrime‐ thoxysilane to convert it into a suitable form.There are various approaches that can be used to immobilize ligands with sulfhydryl groups. The following subsections examine some of these techniques, including the haloacetyl, maleimide, and pyridyl disulfide

Carbonyldiimidazole

N N C

O

<sup>N</sup> <sup>N</sup> <sup>N</sup> <sup>N</sup> <sup>C</sup> O

carboxyl-containing

COOH +

C O

carbamate support

pH 9-10 Ligand - NH2

NH - Ligand

O

C O

**Figure 14.** The carbonyldiimidazole immobilization method [8]

matrix

82 Column Chromatography

**a.** Haloacetyl Method; The haloacetyl method uses supports that contain iodoacetyl or bromoacetyl groups for the immobilization of ligands through sulfhydryl residues. These supports are usually prepared via the reaction of an amine-containing material with iodoacetic or bromoacetic acid in the presence of ethyldimethylaminopropyl carbodii‐ mide (EDC) at pH 4 to 5. EDC reacts with the carboxylic acid in iodo- or bromoacetic acid

methods.

**3.** Hydroxyl-Reactive Methods; A number of methods have been used to couple ligands through hydroxyl groups. However, unlike many amine- and sulfhydryl-reactive methods, techniques for hydroxyl-containing ligands are not that selective. For example, the divinylsulfone method can be used for coupling an amine-, sulfhydryl-, or hydroxylcontaining ligand. Many supports used in affinity chromatography already contain hydroxyl groups on their surface. One way for the activation of these groups is to introduce bisoxirane (epoxy) groups. The most frequently used oxirane for this purpose is 1,4-butanediol diglycidyl ether, which contains two epoxy groups. One of the epoxy groups can react with the hydroxyl groups on a support while the other is used for coupling ligands containing sulfhydryl, amine, the reactivity of the terminal epoxide to other groups follows the order –SH > –NH > –OH. Strong alkaline conditions (pH 11) allow coupling by this method through hydroxyl groups, while amines and sulfhydryl groups can react at a lower pH (pH 7 to 8). Cyanuric chloride is another agent used for attaching a hydroxyl-containing ligand to a support. In this method, this can only be used effectively in the absence of amine groups due to the higher reactivity of these groups. As before‐ mentioned, divinylsulfone can be used for coupling hydroxyl-containing ligands. This, however, is not usually performed if the immobilized ligand is present at a pH higher than 9 to 10.

**•** Carbonyldiimidazole (CDI) method

**•** Tresyl chloride/tosyl chloride method

**•** Azalactone method (for Emphaze supports)

**•** Azalactone method (for Emphaze supports)

**•** Ethyl dimethylaminopropyl carbodiimide (EDC)

**•** Cyanuric chloride method

**•** Epoxy (bisoxirane) method

**•** Divinylsulfone (DVS)

**•** method

Sulfhydryl groups:

**•** Divinylsulfone method

**•** Maleimide method

**•** TNB-thiol method

Hydroxyl groups:

Aldehyde groups

Carboxyl groups

**1.4. Elution**

**•** Hydrazide method

**•** Epoxy (bisoxirane) method

**•** Pyridyl disulfide method

**•** Cyanuric chloride method

**•** Epoxy (bisoxirane) method

**•** Divinylsulfone method

**•** Iodoacetyl/bromoacetyl method

**•** Tresyl chloride/tosyl chloride method

Elution is one of the critical step for successful separation. Sample application in affinity chromatography is performed usually by injection or application in the presence of mobile phase which is prepared in appropriate pH, ionic strength and solvent composition for soluteligand binding. This solvent is referred as application buffer [8]. In the presence of application buffer, compounds which are complementary to the affinity ligand will bind while the other solutes in the sample will tend to pass through the column as nonretained compounds. After

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Activation methods which are used in affinity chromatography can be summarized as follow: Amine groups :


mentioned, divinylsulfone can be used for coupling hydroxyl-containing ligands. This, however, is not usually performed if the immobilized ligand is present at a pH higher

**4.** Carbonyl-Reactive Methods; Although most immobilization techniques involve coupling ligands through amine or sulfhydryl groups, the large number of such groups can create a problem with improper orientation or multipoint attachment. This can be avoided by using alternative groups that occur only in specific locations on the ligand. One example is the immobilization of antibodies through their carbohydrate residues. To use the carbohydrate groups of an antibody (or any other glycoprotein) for immobilization, these groups must first be oxidized to form reactive aldehyde groups. This can be accomplished by enzymatic treatment; however, it is usually performed through mild treatment with periodate. These aldehyde groups are then reacted with a support containing amine or hydrazide groups for ligand immobilization. This approach has been used not only for antibodies but also for glycoenzymes, RNA, and sugars. Supports with amine groups can be used for coupling aldehyde-containing ligands by reductive amination. Hydrazideactivated supports can also be employed for immobilizing ligands with aldehyde groups. Such supports can be prepared by formation aldehyde groups on the support and reaction of these with an excess of a dihydrazide (e.g., oxalic or adipic dihydrazide). An advantage of using a hydrazide-activated support is that no reducing agent is needed to stabilize the

linkage between the ligand and support, as is required in reductive amination.

**5.** Carboxyl-Reactive Methods; There are currently no activated supports that react specifically with a ligand containing carboxyl groups. This is a result of the low nucleo‐ philicity of carboxyl groups in an aqueous solution. However, there are reagents that will react with carboxylic acids and allow them to be activated for ligand attachment. 1- Ethyl-3-(dimethylaminopropyl) carbodiimide is an example of such a reagent. One problem of this process is that severe cross-linking is possible, since amine groups as well as carboxyl groups can react if excess EDC is present. In addition, the activated derivative formed, O-acylisourea, is not stable in an aqueous environment. This means that the activated ligand must be used immediately for immobilization without further pu‐

**6.** Other Immobilization Techniques; Along with noncovalent and covalent immobilization methods, other techniques have been developed for the preperation of affinity supports. Such methods include entrapment, molecular imprinting, and the use of the ligands as both the support and stationary phase. Although these methods are not as common as the approaches already examined, they have important advantages in some applications [8].

Activation methods which are used in affinity chromatography can be summarized as follow:

than 9 to 10.

84 Column Chromatography

rification.

Amine groups :

**•** Cyanogen bromide (CNBr) method

**•** Schiff base (reductive amination) method **•** N-hydroxysuccinimide (NHS) method

**•** Tresyl chloride/tosyl chloride method

Sulfhydryl groups:


Hydroxyl groups:


Aldehyde groups

**•** Hydrazide method

Carboxyl groups

### **1.4. Elution**

Elution is one of the critical step for successful separation. Sample application in affinity chromatography is performed usually by injection or application in the presence of mobile phase which is prepared in appropriate pH, ionic strength and solvent composition for soluteligand binding. This solvent is referred as application buffer [8]. In the presence of application buffer, compounds which are complementary to the affinity ligand will bind while the other solutes in the sample will tend to pass through the column as nonretained compounds. After

all nonretained components are washed off the column, the retained solute or together with ligand as solute-ligand complex can be eluted by applying a solvent. This solvent which is referred as elution buffer represents the strong mobile phase for the column. Later all the interest solutes are eluted from the column, regeneration is performed by elution with application buffer and the column is allowed to regenerate prior to the next sample application [4,8,21]. Step gradient elution or in other word on/off elution method is the most common method employed for affinity chromatography. Figure 15 shows the typical separation in affinity using step gradient elution.

**Figure 15.** Typical separation in affinity using step gradient elution.

Step elution mode is employed if the ligans have high affinity for the target molecule. It is also possible to use isocratic elution in affinity chromatography. This elution mode generally selected if the target molecule and ligand have weak interaction. This approach is known as **Weak Affinity Chromatography** or **Dynamic Affinity Chromatography** [8,21]*.*

In affinity chromatography there are many factors such as strength of solute-ligand interaction, the amount of immobilized ligand present and the kinetics of solute-ligand association and dissociation which have important influences on retention and elution of the compound. The reaction between the target protein (T) and ligand (L) on the other word binding (adsorption) and elution (desorption) process can be explain by following equation in case of a target protein has single site binding to a ligand [1,4,21]*.*

18

[LT] is the concentration of the ligand/target complex

L0 is the concentration of ligand (usually 10-4 - 10-2M)

*K*D+L0

KD is the equilibrium dissociation constant

Bound target Total target <sup>≈</sup> *<sup>L</sup>* <sup>0</sup>

achieve successful binding the ratio should be near 1 in this equation.

The equation that is placed below explains the bound target-total target ratio. In order to

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KD can be changed by pH, ionic strength, temperature and other parameters. Therefore these parameters can be used to control the binding and elution efficiency of the reaction [1,4,22]

Obtaining stable biomolecules in high yield and purity is aimed for elution process. Elution is achieved by reducing the association constant of the ligand-solute interaction. Biospecific or non-specific elution can be utilized. Biospecific elution is based on solute displacement from the column by addition of molecule that acts as a competing agent. Two different types of biospecific elution can be applied for elution. In first method, normal role elution, molecule competes with the ligand for binding the desired solute. In second type of biospecific elution, reversed role elution, molecule competes with desired solute for binding the ligand [13]. The main advantage of biospecific elution is that a target can be gently removed from the column. However this elution is slow and generally results in broad solute peaks. Additionally competing agent needs to be removed from the eluted solvent therefore usage is limited. Another disadvantage especially in analytical application is need to use a competing agent that does not produce a large background signal under the conditions used for analyte detection [8]. Non-specific elution is performed by changing solvent conditions like pH, ionic strength and polarity. High concentration of chaotropic salts (NaCl, MgCl2 or LiCl), denatu‐ rating agents and detergents (guanidine hydrochloride, sodium dodecyl sulfate and urea) can be used. Organic solvents can be used especially for the elution of low molecular weight compounds [13]. Alteration in structure of the solute or ligand which leads to a lower associ‐ ation constant and lower solute retention is provided by nonspecific elution [8]. Non-specific elution is faster than specific elution but there is a risk for denaturation of solute. The conditions which are applied for the elution may be too hard for column. If this is not considered it may

result in long column regeneration times or irreversible loss of ligand activity [8].

For biospecific elution solvent is selected according to the type of target and ligand. The solvent usually has a pH and ionic composition similar to the application buffer but contains a competing agent. Reversed role elution is generally preferred when the target is a small compound while the normal elution is often used for isolation of macromolecules. Readily available in an inexpensive form and be soluble in the elution buffer are desired properties for competing agent in reverse role elution. In reversed-role elution it must be possible to remove the competing agent from the target when the affinity column is used for purification [8].

A wide range of mobile phase additives can be used in non-specific elution. In this elution nature of the target-ligand interaction is changed. This can be achieved by several ways such


[LT] is the concentration of the ligand/target complex

The equation that is placed below explains the bound target-total target ratio. In order to achieve successful binding the ratio should be near 1 in this equation.

Bound target Total target <sup>≈</sup> *<sup>L</sup>* <sup>0</sup> *K*D+L0

18

Time

all nonretained components are washed off the column, the retained solute or together with ligand as solute-ligand complex can be eluted by applying a solvent. This solvent which is referred as elution buffer represents the strong mobile phase for the column. Later all the interest solutes are eluted from the column, regeneration is performed by elution with application buffer and the column is allowed to regenerate prior to the next sample application [4,8,21]. Step gradient elution or in other word on/off elution method is the most common method employed for affinity chromatography. Figure 15 shows the typical separation in

L + T LT

[L][T] [LT]

KD =

Binding

Elution

Response

Nonretained peak retained peak

Step elution mode is employed if the ligans have high affinity for the target molecule. It is also possible to use isocratic elution in affinity chromatography. This elution mode generally selected if the target molecule and ligand have weak interaction. This approach is known as

In affinity chromatography there are many factors such as strength of solute-ligand interaction, the amount of immobilized ligand present and the kinetics of solute-ligand association and dissociation which have important influences on retention and elution of the compound. The reaction between the target protein (T) and ligand (L) on the other word binding (adsorption) and elution (desorption) process can be explain by following equation in case of a target protein

[T] is the concentration of free target <sup>19</sup>

**Weak Affinity Chromatography** or **Dynamic Affinity Chromatography** [8,21]*.*

affinity using step gradient elution.

Application buffer

86 Column Chromatography

Elution buffer

has single site binding to a ligand [1,4,21]*.*

KD is the equilibrium dissociation constant

[L] is the concentration of free ligand

**Figure 15.** Typical separation in affinity using step gradient elution.

L0 is the concentration of ligand (usually 10-4 - 10-2M)

KD is the equilibrium dissociation constant

KD can be changed by pH, ionic strength, temperature and other parameters. Therefore these parameters can be used to control the binding and elution efficiency of the reaction [1,4,22]

Obtaining stable biomolecules in high yield and purity is aimed for elution process. Elution is achieved by reducing the association constant of the ligand-solute interaction. Biospecific or non-specific elution can be utilized. Biospecific elution is based on solute displacement from the column by addition of molecule that acts as a competing agent. Two different types of biospecific elution can be applied for elution. In first method, normal role elution, molecule competes with the ligand for binding the desired solute. In second type of biospecific elution, reversed role elution, molecule competes with desired solute for binding the ligand [13]. The main advantage of biospecific elution is that a target can be gently removed from the column. However this elution is slow and generally results in broad solute peaks. Additionally competing agent needs to be removed from the eluted solvent therefore usage is limited. Another disadvantage especially in analytical application is need to use a competing agent that does not produce a large background signal under the conditions used for analyte detection [8]. Non-specific elution is performed by changing solvent conditions like pH, ionic strength and polarity. High concentration of chaotropic salts (NaCl, MgCl2 or LiCl), denatu‐ rating agents and detergents (guanidine hydrochloride, sodium dodecyl sulfate and urea) can be used. Organic solvents can be used especially for the elution of low molecular weight compounds [13]. Alteration in structure of the solute or ligand which leads to a lower associ‐ ation constant and lower solute retention is provided by nonspecific elution [8]. Non-specific elution is faster than specific elution but there is a risk for denaturation of solute. The conditions which are applied for the elution may be too hard for column. If this is not considered it may result in long column regeneration times or irreversible loss of ligand activity [8].

For biospecific elution solvent is selected according to the type of target and ligand. The solvent usually has a pH and ionic composition similar to the application buffer but contains a competing agent. Reversed role elution is generally preferred when the target is a small compound while the normal elution is often used for isolation of macromolecules. Readily available in an inexpensive form and be soluble in the elution buffer are desired properties for competing agent in reverse role elution. In reversed-role elution it must be possible to remove the competing agent from the target when the affinity column is used for purification [8].

A wide range of mobile phase additives can be used in non-specific elution. In this elution nature of the target-ligand interaction is changed. This can be achieved by several ways such 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 substances [8].

Columns, dialysis membranes, capillaries or beads may be used in immunoaffinity application which is a non-covalent, irreversible purification process based on highly specific interactions

Affinity Chromatography and Importance in Drug Discovery

http://dx.doi.org/10.5772/55781

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Initially, the antibodies should be purified prior to prepare the immunoaffinity column. Precipitation with ammonium sulfate, ion-exchange chromatography, gel filtration chraoma‐ tography or affinity chromatography may be employed with the aim of antibody purification. Activated beads which are coated with bacterial proteins A or G may be used as the support material. Some parameters may be changed for the elution of the sample solution for example the ionic conditions of mobile phase may be changed or chaotropic buffers may be used [11].

Both small and large analytes can be determined using direct detection in IAC. Additionally it is possible to use this technique either separately or in combination with other chromato‐ graphic techniques [1]. If this technique is performed as part of HPLC system the method can

Immunoaffinity chromatography is probably the most highly specific of all forms of bioaffinity chromatography. However this technique has some disadvantages such as: this technique relatively high cost, leakage of ligands may accur from the column and sometimes the

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

Lectins which are non-immun proteins are produced by plants, vertebrates and invertebrates. Especially various plant seeds synthesize high levels of lectins [24]. Certain types of carbohy‐ drate residues may be seperated via this method due all lectins have the ability to recognize and bind these types of compounds. Mostly used lectins for affinity columns are concanavalin A, soybean lectin and wheat germ agglutinin [1, 24]. Concanavalin A is specific for α-Dmannose and α-D-glucose residues while wheat germ agglutinin binds to D-*N*-acetylglucosamine. Lectins which are commonly used for the isolation of compunds containing carbohydrates such as polysaccharides, glycoproteins and glycolipids in affinity chromatog‐

be referred as high performance immunoaffinity chromatography.

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

chromatography is needed [1].

raphy are given in Table 4. [1].

**2.3. Lectin affinity chromatography**

desorption procedure results in partial denaturation of the bound protein [24].

between analyte and antibody [11].
