**1. Introduction**

[29] Ge Y, Duan Y, Fang G, Zhang Y, Wang S. Polysaccharides from fruit calyx of Physa‐ lis alkekengi var. francheti Isolation, purification, structural features and antioxidant

[30] Chen R, meng F, Liu Z, Chen R, Zhang M. Antitumor activities of different fractions of polysaccharide purified from *Ornithogalum caudatum* Ait. Carbohydrate Polymers

[31] Battestin V, Macedo GA. Effects of temperature, pH and additives on the activity of tannase produced by Paecilomyces variottii. Electronic Journal of Biotechnology 2007;10 http://www.ejbiotechnology.cl/ content/vol10/issue2/full/9/index.html#6 (ac‐

[32] Kato A, Kano E, Adachi I, Molyneux RJ, Watson AA, Nash RJ, Fleet GWJ, Wormald MR, Kizu H, Ikeda K, Asano N. Australine and related alkaloids: easy structural con‐ firmation by 13C NMR spectral data and biological activities. Tetrahedron 2003;14

[33] Sakamoto S, Hatakeyama M, Ito T, Handa H. Tools and methodologies capable of isolating and identifying a target molecule for a bioactive compound. Bioorganic and

[34] Dragull K, Beck JJ. Isolation of Natural Products by Ion Exchange Methods. Methods

[35] Dufresne C. Isolation by Ion Exchange Methods. In Natural Products Isolation Con‐

activities. Carbohydrate Polymers 2009;77 188-193.

Medicinal Chemistry 2012; 20 1990-2001.

in Molecular Biology 2012; 864 189-219.

nell RJP. New Jersey: Humana Press; 1998.

2010;80 845-851.

58 Column Chromatography

cesses 20.09.2012).

325-331.

Affinity chromatography which is known as a liquid chromatographic technique for separation and analysis of biomolecules based on their biological functions or individual structures has become increasingly important and useful separation method in pharmaceut‐ ical science, biochemistry, biotechnology and environmental science in recent years [1]. This technique is especially known as the most specific and effective technique for protein purification [2]. Separation of the biomolecules is based on highly specific biological interactions between two molecules, such as enzyme and substrate. These interactions, which are typically reversible, are used for purification by placing one of the interacting molecules, referred to as affinity ligand, onto a solid matrix to create a stationary phase while the target molecule is in the mobile phase [3]. Any component can be used as a ligand to purify its respective binding partner. Some typical biological interactions, frequently used in affinity chromatography, can be given as;


**•** Metal ions ↔ . Poly (His) fusion proteins, native proteins with histidine, cysteine and/or tryptophan residues on their surface [4-5].

nonretained compounds. After all nonretained components are washed off the column, binding solute or together with ligand as solute-ligand complex are eluted by applying a solvent. This solvent which is referred as elution buffer represents the strong mobile phase for the column. Later all the interested solutes are eluted from the column, then application buffer is applied and the column is allowed to regenerate prior to the next sample application [4,8].

> Sample is applied under optimum conditions that favor specific binding of the target molecule(s) to complementary binding molecules (the ligand). Desired molecules bind specifically, but reversibly, to the ligand and unbound

Target protein is recovered by changing conditions to favor elution of the bound molecules. Elution is performed specifically using a competitive ligand, or non‐specifically, by changing the pH, ionic strength or polarity. Target protein is

Affinity medium is re‐equilibrated with binding buffer

Affinity medium is equilibrated with binding buffer

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61

material is washed through the column.

collected in a purified, concentrated form.

.

.

Figure 2, a typical scheme of an affinity chromatography application is shown.

The conditions in which the sample is applied to the column are chosen considering the conditions which the interaction between analyte and ligand is strong, mostly resembling the natural conditions of the analyte and ligand. The content apart from the analyte passes through the column without or with weak binding to the ligand while the analyte is retarded. After the analyte is obtained generally by using an elution buffer, the column is regenerated by washing with the application buffer in order to prepare the column for the next injection [1]. In the

As it is defined above; this technique is based on the interactions between specific bioactive substances, so the ligands are supposed to be originally biological substances, nevertheless columns with nonbiological ligands are also available and the same term "affinity chroma‐ tography" is used for the techniques performed by using these ligands. In order to distinguish the techniques according to the origin of the ligand, affinity chromatography with biological ligands may be termed as "bioaffinity chromatography" or "biospesific adsorbtion" [1]. The wide application potential of affinity chromatography leaded to the development of derived

**Figure 1.** Separation procedure in affinity chromatography

techniques some of which are listed below [7].

**•** Immunoaffinity chromatography

In case a ligand is immobilized on a polymeric carrier, usually by covalent coupling, and filled in a column, it is possible to separate the substances which have affinity to the ligand and the other substances. As the solution containing the biologically active substance applied to the column, the compounds which have no affinity to the insoluble ligand will pass through the column and the biologically active compound will be captured on the column, in favorable conditions. The sorbed compounds can then easily be dissociated by changing the external conditions, such as ionic strength, pH, solvent, temperature etc. or alternatively by using dissociating agents [6-7]. As a result, it is possible to isolate and purify the analyte or make quantitative analysis with a suitable, immobilized ligand by means of molecular recognition [1-2].

Macromolecules such as proteins, polysaccharides, nucleic acids differ only in their physicochemical properties within the individual groups and their isolation on the basis of these differences is therefore difficult and time consuming. Considerable decreases may occur during their isolation procedure due to denaturation, cleavage, enzymatic hydrolysis, etc. The ability to bind other molecules reversibly is one of the most important properties of these molecules. The formation of specific and reversible complexes of biological macromo‐ lecules can serve as basis of their separation, purification and analysis by the affinity chromatography [6].

Affinity chromatography is one of the oldest forms of liquid chromatography method [8]. The first use of the idea of affinity chromatography may be considered as the isolation of α-amylase by using an insoluble substrate, starch, in 1910 by Starkenstein [6,9]. Similar studies with starch and amylase were carried out in the 1920s through 1940s by other investigators. In another study polygalacturonase was used as a support and ligand for the adsorption of alginic acid, the purification of pepsin through the use of edestin, a crystalline protein and the isolation of porcine elastase with powdered elastin were also performed. Afterwards Willstatter et al. enriched lipase by selective adsorption onto powdered stearic aicd [10]. The majority of the previous studies related purification of the enzymes. However the selective purification of antibodies with biological ligands was also being conducted. In 1920, it was reported that antibodies can recognize and bind substances with a specific structure, "antigens" [8]. This principle is firstly used in order to isolate rabbit anti-bovine serum albumin antibodies on a specific immunoadsorbent column consisting bovine serum albumin coupled to diazotized *paminobenzyl*-cellulose [10]. According to this approach, antibodies were isolated using urease and exhibited that these antibodies were proteins [8].

Separation procedure in affinity chromatography can be simply illustrated as shown in Figure 1. A sample containing the compound of interest is applied to the affinity column in the presence of mobile phase which was prepared in suitable pH, ionic strength and solvent composition for solute-ligand binding. This solvent which is referred as the application buffer presents the weak mobile phase of an affinity chromatography. While the sample is passing through the column compounds which are complementary to the affinity ligand will bind. However other solutes in the sample will tend to be washed off or eluted from the column as nonretained compounds. After all nonretained components are washed off the column, binding solute or together with ligand as solute-ligand complex are eluted by applying a solvent. This solvent which is referred as elution buffer represents the strong mobile phase for the column. Later all the interested solutes are eluted from the column, then application buffer is applied and the column is allowed to regenerate prior to the next sample application [4,8].

**•** Metal ions ↔ . Poly (His) fusion proteins, native proteins with histidine, cysteine and/or

In case a ligand is immobilized on a polymeric carrier, usually by covalent coupling, and filled in a column, it is possible to separate the substances which have affinity to the ligand and the other substances. As the solution containing the biologically active substance applied to the column, the compounds which have no affinity to the insoluble ligand will pass through the column and the biologically active compound will be captured on the column, in favorable conditions. The sorbed compounds can then easily be dissociated by changing the external conditions, such as ionic strength, pH, solvent, temperature etc. or alternatively by using dissociating agents [6-7]. As a result, it is possible to isolate and purify the analyte or make quantitative analysis with a suitable, immobilized ligand by

Macromolecules such as proteins, polysaccharides, nucleic acids differ only in their physicochemical properties within the individual groups and their isolation on the basis of these differences is therefore difficult and time consuming. Considerable decreases may occur during their isolation procedure due to denaturation, cleavage, enzymatic hydrolysis, etc. The ability to bind other molecules reversibly is one of the most important properties of these molecules. The formation of specific and reversible complexes of biological macromo‐ lecules can serve as basis of their separation, purification and analysis by the affinity

Affinity chromatography is one of the oldest forms of liquid chromatography method [8]. The first use of the idea of affinity chromatography may be considered as the isolation of α-amylase by using an insoluble substrate, starch, in 1910 by Starkenstein [6,9]. Similar studies with starch and amylase were carried out in the 1920s through 1940s by other investigators. In another study polygalacturonase was used as a support and ligand for the adsorption of alginic acid, the purification of pepsin through the use of edestin, a crystalline protein and the isolation of porcine elastase with powdered elastin were also performed. Afterwards Willstatter et al. enriched lipase by selective adsorption onto powdered stearic aicd [10]. The majority of the previous studies related purification of the enzymes. However the selective purification of antibodies with biological ligands was also being conducted. In 1920, it was reported that antibodies can recognize and bind substances with a specific structure, "antigens" [8]. This principle is firstly used in order to isolate rabbit anti-bovine serum albumin antibodies on a specific immunoadsorbent column consisting bovine serum albumin coupled to diazotized *paminobenzyl*-cellulose [10]. According to this approach, antibodies were isolated using urease

Separation procedure in affinity chromatography can be simply illustrated as shown in Figure 1. A sample containing the compound of interest is applied to the affinity column in the presence of mobile phase which was prepared in suitable pH, ionic strength and solvent composition for solute-ligand binding. This solvent which is referred as the application buffer presents the weak mobile phase of an affinity chromatography. While the sample is passing through the column compounds which are complementary to the affinity ligand will bind. However other solutes in the sample will tend to be washed off or eluted from the column as

tryptophan residues on their surface [4-5].

means of molecular recognition [1-2].

and exhibited that these antibodies were proteins [8].

chromatography [6].

60 Column Chromatography

Affinity medium is equilibrated with binding buffer

Sample is applied under optimum conditions that favor specific binding of the target molecule(s) to complementary binding molecules (the ligand). Desired molecules bind specifically, but reversibly, to the ligand and unbound material is washed through the column.

Target protein is recovered by changing conditions to favor elution of the bound molecules. Elution is performed specifically using a competitive ligand, or non‐specifically, by changing the pH, ionic strength or polarity. Target protein is collected in a purified, concentrated form.

Affinity medium is re‐equilibrated with binding buffer

**Figure 1.** Separation procedure in affinity chromatography

The conditions in which the sample is applied to the column are chosen considering the conditions which the interaction between analyte and ligand is strong, mostly resembling the natural conditions of the analyte and ligand. The content apart from the analyte passes through the column without or with weak binding to the ligand while the analyte is retarded. After the analyte is obtained generally by using an elution buffer, the column is regenerated by washing with the application buffer in order to prepare the column for the next injection [1]. In the Figure 2, a typical scheme of an affinity chromatography application is shown.

As it is defined above; this technique is based on the interactions between specific bioactive substances, so the ligands are supposed to be originally biological substances, nevertheless columns with nonbiological ligands are also available and the same term "affinity chroma‐ tography" is used for the techniques performed by using these ligands. In order to distinguish the techniques according to the origin of the ligand, affinity chromatography with biological ligands may be termed as "bioaffinity chromatography" or "biospesific adsorbtion" [1]. The wide application potential of affinity chromatography leaded to the development of derived techniques some of which are listed below [7].

**•** Immunoaffinity chromatography

**•** Avidin-biotin immobilized system

**•** Affinity repulsion chromatography **•** Perfusion affinity chromatography

**•** Affinity tails chromatography

**•** Theophilic chromatography

**•** Weak affinity chromatography

**•** Molecular imprinting affinity

**•** Receptor affinity chromatography

**•** Membrane-based affinity chromatography

Affinity chromatography utilizes specific and irreversible biological interactions between a ligand covalently coupled to a support material and its complementary target. The solid support and ligand covalently attached on it, selectively adsorbs the complementary substance from the sample. The unbound part of the sample is removed and the purified substance can easily be recovered [11]. Selectivity of the ligand, recovery process, throughput, reproducibil‐ ity, stability and economical criteria are some of the factors that influence the success of affinity chromatography process [9]. Successful affinity purification requires a certain degree of knowledge and understanding of the nature of interactions between the target molecule and the ligand to help determine the selection of an appropriate affinity ligand and purification procedure [3]. Therefore prior to start the process, materials and specifications listed below

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63

For successful separation in affinity chromatography, the important parameter is that solute of interest should be bound firmly and specifically while leaving all other molecules. This requires that the support within the column contain an affinity ligand that is capable of forming a suitably strong complex with the solute of interest [8]. The other important property is that the, support material must be biologically and chemically inert to avoid

**•** Metal-chelate affinity chromatography

**•** Covalent affinity chromatography

**•** Hydrophobic chromatography

need to be selected [11]:

**•** Immobilization method

**1.1. Support material**

**•** Conditions for adsorption and desorption

**•** Support material **•** Activation method

**•** Ligand

**Figure 2.** An example of a typical scheme of an affinity chromatography application [1].


Affinity chromatography utilizes specific and irreversible biological interactions between a ligand covalently coupled to a support material and its complementary target. The solid support and ligand covalently attached on it, selectively adsorbs the complementary substance from the sample. The unbound part of the sample is removed and the purified substance can easily be recovered [11]. Selectivity of the ligand, recovery process, throughput, reproducibil‐ ity, stability and economical criteria are some of the factors that influence the success of affinity chromatography process [9]. Successful affinity purification requires a certain degree of knowledge and understanding of the nature of interactions between the target molecule and the ligand to help determine the selection of an appropriate affinity ligand and purification procedure [3]. Therefore prior to start the process, materials and specifications listed below need to be selected [11]:


**•** High performance affinity chromatography

**Figure 2.** An example of a typical scheme of an affinity chromatography application [1].

**•** Affinity density perturbation

**•** Affinity partitioning

62 Column Chromatography

**•** Affinity electrophoresis

**•** Affinity precipitation

**•** Library-derived affinity ligands **•** Lectin affinity chromatography

**•** Dye-ligand affinity chromatography

**•** Centrifuged affinity chromatography **•** Filter affinity transfer chromatography

**•** Affinity capillay electrophoresis


### **1.1. Support material**

For successful separation in affinity chromatography, the important parameter is that solute of interest should be bound firmly and specifically while leaving all other molecules. This requires that the support within the column contain an affinity ligand that is capable of forming a suitably strong complex with the solute of interest [8]. The other important property is that the, support material must be biologically and chemically inert to avoid

the unspecific bindings [8,11] which requires that the support has a chemical character that is very similar to that of the medium in which it is operating. Since almost all affinity separations occur in aqueous solutions, the support should thus be as hydrophilic as possible. As a rule, the mobile phase used in affinity separations has a low ionic strength. The support should therefore contain as few charges as possible to prevent ionic interac‐ tions. Many supports are available which have desired properties or they gain such characteristics by hydrophilic coating [8]. Generally solid materials are used as support material though some soluble macromolecular materials are sometimes preferred for twophase aqueous affinity partition processes. Uniformity in particle size and ease of the activation process are also required for support material that is used in affinity chromatog‐ raphy applications [11]. For the affinity chromatography at low pressure, nonrigid gels with large particle size are generally used as support materials while materials with small, rigid particles or synthetic polymers which are stable under high pressure and flow rates are used in high performance affinity applications [8,12].

> Sephacryl (allyldextran cross-linked by *N,*N'-methylenebisacrylamide) are the two types of dextran gel used in separation. Sephadex is mainly used as a glucose polymer and employed for purification of many molecules such as lectins from *Helix pomatia* and *Vicia faba*, exoamylase

(a) (b)

material that is used in affinity chromatography applications [11]. For the affinity chromatography at low pressure, nonrigid gels with large particle size are generally used as support materials while materials with small, rigid particles or synthetic polymers

There are many commercially available support materials for affinity chromatography can be divided into three groups as; natural (agarose, dextrose, cellulose); synthetic (acrylamide, polystyrene, polymethylacrylate) and inorganic (silica, glass) materials [7,13]. The most popular support material is agarose [13]. Agarose was used in the first modern application of affinity chromatography and still the most commonly preferred one [8]. Agarose consists of alternatively linked 1,3 bound β-D- galactopyranose and 1,4 bound 3,6-anhydro-α-*L*-galactopyranose, as shown in **Figure 3** [8,14]. Agarose gels are stable to eluants with high concentrations of salt, urea, guanidine hydrochloride, detergents or water-miscible organic solvents but its stability is less beyond pH 4-9. To increase the thermal and chemical stability, cross-linked agarose is prepared. Cross-linked agarose is commercially available (Sepharose) and it can be used with many solvents, over pH 3-14 and at high temperatures up to 70°C [11]. However, strong acids, oxidizers as well as some rare enzymes may be harmful to agarose due to their damaging effects. On the other hand mild acid hydrolysis increases the quantity of sterically available galactose residues and turns agarose into an excellent sorbent for galactose binding proteins [14]. Due to its large beads and macroporous, accessible pore structures, agarose is well designed for use with large molecules. High capacity, presence of functional groups, good chemical stability especially at high pH, low non-spesific binding and good reproducibility are the advantage of agarose. Some properties of agarose such as solubility in hot water and non-aqueous solutions, sensitivity to microbial degredation and lack of rigidity restrict the usage under low or medium pressure [15]. Furthermore agarose must not be frozen or air dried. It is sold under several trade names, including Sepharose Fast Flow or

Cellulose is another example of polysaccharides which is used as support in affinity chromatography. Cellulose has a historical significance. Phospo- and DNA-cellulose are used especially for DNA related separations [14]. Antibody and enzyme purifications have also been carried out. However its fibrous and non-uniform character limits its use since cellulose detains macromolecules

Dextran which is a linear α-1,6-linked glucose polymer produced by *Leuconostoc mesenteroides* is also used in affinity chromatography. Sephadex (cross-linked by glyceryl bridges) and Sephacryl (allyldextran cross-linked by *N,*N' methylenebisacrylamide) are the two types of dextran gel used in separation. Sephadex is mainly used as a glucose polymer and employed for purification of many molecules such as lectins from *Helix pomatia* and *Vicia faba*, exoamylase from *Pseudomonas* 

Polystyrene (**Figure 4**), which is a polymeric support is also unsuitable in its original form for affinity separations due to the highly hydrophobic character. Native polystyrene, which is often used as a reversed-phase material, must be first rendered hydrophilic by

which are stable under high pressure and flow rates are used in high performance affinity applications [8,12].

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Polystyrene (Figure 4), which is a polymeric support is also unsuitable in its original form for affinity separations due to the highly hydrophobic character. Native polystyrene, which is often used as a reversed-phase material, must be first rendered hydrophilic by one of various

(a) (b)

one of various surface-coating techniques before used in other chromatographic methods [8].

(a) (b)

Polymeric supports based on polyacrylamide are synthesized by copolymerization of acryla‐ mide and a cross-linking reagent and can be used directly in affinity chromatography due to its more hydrophilic properties than polystyrene supports. Polyacrylamide gels are either soft

**Figure 4. (a)** Structure of polystyrene divinilybenzene **(b)** SEM image of polystyrene beads(x500) [17]

CH CH2 CH CH2 CH CH2 CH <sup>2</sup> CH

surface-coating techniques before used in other chromatographic methods [8].

Figure 3. **(a).** Structure of agarose **(b).** SEM image of agarose [16]

H

HO O

H O

CH2

H

O

H

from *Pseudomonas stutzeri* [14].

O

CH2OH

H

H

H

OH

O

**Figure 3. (a).** Structure of agarose **(b).** SEM image of agarose [16]

H

[11].

*stutzeri* [14].

Affi-Gel.

O

H

HO

CH2 CH CH2 CH CH2

CH CH2 CH CH2 CH CH2

CH CH2 CH CH2 CH CH2 CH <sup>2</sup> CH

CH2 CH CH2 CH CH2

CH CH2 CH CH2 CH <sup>2</sup> CH

There are many commercially available support materials for affinity chromatography can be divided into three groups as; natural (agarose, dextrose, cellulose); synthetic (acrylamide, polystyrene, polymethylacrylate) and inorganic (silica, glass) materials [7,13]. The most popular support material is agarose [13]. Agarose was used in the first modern application of affinity chromatography and still the most commonly preferred one [8]. Agarose consists of alternatively linked 1,3 bound β-D- galactopyranose and 1,4 bound 3,6-anhydro-α-*L*-galacto‐ pyranose, as shown in Figure 3 [8,14]. Agarose gels are stable to eluants with high concentra‐ tions of salt, urea, guanidine hydrochloride, detergents or water-miscible organic solvents but its stability is less beyond pH 4-9. To increase the thermal and chemical stability, cross-linked agarose is prepared. Cross-linked agarose is commercially available (Sepharose) and it can be used with many solvents, over pH 3-14 and at high temperatures up to 70°C [11]. However, strong acids, oxidizers as well as some rare enzymes may be harmful to agarose due to their damaging effects. On the other hand mild acid hydrolysis increases the quantity of sterically available galactose residues and turns agarose into an excellent sorbent for galactose binding proteins [14]. Due to its large beads and macroporous, accessible pore structures, agarose is well designed for use with large molecules. High capacity, presence of functional groups, good chemical stability especially at high pH, low non-spesific binding and good reproducibility are the advantage of agarose. Some properties of agarose such as solubility in hot water and non-aqueous solutions, sensitivity to microbial degredation and lack of rigidity restrict the usage under low or medium pressure [15]. Furthermore agarose must not be frozen or air dried. It is sold under several trade names, including Sepharose Fast Flow or Affi-Gel.

Cellulose is another example of polysaccharides which is used as support in affinity chroma‐ tography. Cellulose has a historical significance. Phospo- and DNA-cellulose are used especially for DNA related separations [14]. Antibody and enzyme purifications have also been carried out. However its fibrous and non-uniform character limits its use since cellulose detains macromolecules [11].

Dextran which is a linear α-1,6-linked glucose polymer produced by *Leuconostoc mesenter‐ oides* is also used in affinity chromatography. Sephadex (cross-linked by glyceryl bridges) and methylenebisacrylamide) are the two types of dextran gel used in separation. Sephadex is mainly used as a glucose polymer and

material that is used in affinity chromatography applications [11]. For the affinity chromatography at low pressure, nonrigid gels with large particle size are generally used as support materials while materials with small, rigid particles or synthetic polymers

There are many commercially available support materials for affinity chromatography can be divided into three groups as; natural (agarose, dextrose, cellulose); synthetic (acrylamide, polystyrene, polymethylacrylate) and inorganic (silica, glass) materials [7,13]. The most popular support material is agarose [13]. Agarose was used in the first modern application of affinity chromatography and still the most commonly preferred one [8]. Agarose consists of alternatively linked 1,3 bound β-D- galactopyranose and 1,4 bound 3,6-anhydro-α-*L*-galactopyranose, as shown in **Figure 3** [8,14]. Agarose gels are stable to eluants with high concentrations of salt, urea, guanidine hydrochloride, detergents or water-miscible organic solvents but its stability is less beyond pH 4-9. To increase the thermal and chemical stability, cross-linked agarose is prepared. Cross-linked agarose is commercially available (Sepharose) and it can be used with many solvents, over pH 3-14 and at high temperatures up to 70°C [11]. However, strong acids, oxidizers as well as some rare enzymes may be harmful to agarose due to their damaging effects. On the other hand mild acid hydrolysis increases the quantity of sterically available galactose residues and turns agarose into an excellent sorbent for galactose binding proteins [14]. Due to its large beads and macroporous, accessible pore structures, agarose is well designed for use with large molecules. High capacity, presence of functional groups, good chemical stability especially at high pH, low non-spesific binding and good reproducibility are the advantage of agarose. Some properties of agarose such as solubility in hot water and non-aqueous solutions, sensitivity to microbial degredation and lack of rigidity restrict the usage under low or medium pressure

which are stable under high pressure and flow rates are used in high performance affinity applications [8,12].

Figure 3. **(a).** Structure of agarose **(b).** SEM image of agarose [16] **Figure 3. (a).** Structure of agarose **(b).** SEM image of agarose [16]

Affi-Gel.

the unspecific bindings [8,11] which requires that the support has a chemical character that is very similar to that of the medium in which it is operating. Since almost all affinity separations occur in aqueous solutions, the support should thus be as hydrophilic as possible. As a rule, the mobile phase used in affinity separations has a low ionic strength. The support should therefore contain as few charges as possible to prevent ionic interac‐ tions. Many supports are available which have desired properties or they gain such characteristics by hydrophilic coating [8]. Generally solid materials are used as support material though some soluble macromolecular materials are sometimes preferred for twophase aqueous affinity partition processes. Uniformity in particle size and ease of the activation process are also required for support material that is used in affinity chromatog‐ raphy applications [11]. For the affinity chromatography at low pressure, nonrigid gels with large particle size are generally used as support materials while materials with small, rigid particles or synthetic polymers which are stable under high pressure and flow rates are

There are many commercially available support materials for affinity chromatography can be divided into three groups as; natural (agarose, dextrose, cellulose); synthetic (acrylamide, polystyrene, polymethylacrylate) and inorganic (silica, glass) materials [7,13]. The most popular support material is agarose [13]. Agarose was used in the first modern application of affinity chromatography and still the most commonly preferred one [8]. Agarose consists of alternatively linked 1,3 bound β-D- galactopyranose and 1,4 bound 3,6-anhydro-α-*L*-galacto‐ pyranose, as shown in Figure 3 [8,14]. Agarose gels are stable to eluants with high concentra‐ tions of salt, urea, guanidine hydrochloride, detergents or water-miscible organic solvents but its stability is less beyond pH 4-9. To increase the thermal and chemical stability, cross-linked agarose is prepared. Cross-linked agarose is commercially available (Sepharose) and it can be used with many solvents, over pH 3-14 and at high temperatures up to 70°C [11]. However, strong acids, oxidizers as well as some rare enzymes may be harmful to agarose due to their damaging effects. On the other hand mild acid hydrolysis increases the quantity of sterically available galactose residues and turns agarose into an excellent sorbent for galactose binding proteins [14]. Due to its large beads and macroporous, accessible pore structures, agarose is well designed for use with large molecules. High capacity, presence of functional groups, good chemical stability especially at high pH, low non-spesific binding and good reproducibility are the advantage of agarose. Some properties of agarose such as solubility in hot water and non-aqueous solutions, sensitivity to microbial degredation and lack of rigidity restrict the usage under low or medium pressure [15]. Furthermore agarose must not be frozen or air dried. It is sold under several trade names, including Sepharose Fast Flow or Affi-Gel.

Cellulose is another example of polysaccharides which is used as support in affinity chroma‐ tography. Cellulose has a historical significance. Phospo- and DNA-cellulose are used especially for DNA related separations [14]. Antibody and enzyme purifications have also been carried out. However its fibrous and non-uniform character limits its use since cellulose detains

Dextran which is a linear α-1,6-linked glucose polymer produced by *Leuconostoc mesenter‐ oides* is also used in affinity chromatography. Sephadex (cross-linked by glyceryl bridges) and

used in high performance affinity applications [8,12].

64 Column Chromatography

macromolecules [11].

Sephacryl (allyldextran cross-linked by *N,*N'-methylenebisacrylamide) are the two types of dextran gel used in separation. Sephadex is mainly used as a glucose polymer and employed for purification of many molecules such as lectins from *Helix pomatia* and *Vicia faba*, exoamylase from *Pseudomonas stutzeri* [14]. Cellulose is another example of polysaccharides which is used as support in affinity chromatography. Cellulose has a historical significance. Phospo- and DNA-cellulose are used especially for DNA related separations [14]. Antibody and enzyme purifications have also been carried out. However its fibrous and non-uniform character limits its use since cellulose detains macromolecules

Polystyrene (Figure 4), which is a polymeric support is also unsuitable in its original form for affinity separations due to the highly hydrophobic character. Native polystyrene, which is often used as a reversed-phase material, must be first rendered hydrophilic by one of various surface-coating techniques before used in other chromatographic methods [8]. [11]. Dextran which is a linear α-1,6-linked glucose polymer produced by *Leuconostoc mesenteroides* is also used in affinity chromatography. Sephadex (cross-linked by glyceryl bridges) and Sephacryl (allyldextran cross-linked by *N,*N'-

**Figure 4. (a)** Structure of polystyrene divinilybenzene **(b)** SEM image of polystyrene beads(x500) [17]

Polymeric supports based on polyacrylamide are synthesized by copolymerization of acryla‐ mide and a cross-linking reagent and can be used directly in affinity chromatography due to its more hydrophilic properties than polystyrene supports. Polyacrylamide gels are either soft CH CH2 CH CH2 CH <sup>2</sup> CH

(a) (b)

or have small pores. However this gel is resistant against enzymatic attacks and does not absorb biomolecules, thus it is used widely despite its mechanic disadvantages [8,14].

ratio *Rs/Rp,* one finds that the pore diameter should be at least five times the diameter of the

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67

For a protein in normal size (i.e., a diameter around 60 Å), a ratio of five for *Rp/Rs* means that the support pores should be in the range of 300 Å. Several common supports are available with such pore sizes. Support materials with very large pores give essentially unhindered diffusion for most solutes, but they also have a smaller surface area per milliliter of bed volume than supports with smaller pores. This reduced surface area leads to a diminished binding capacity. As a rule, a pore size of 300 to 700 Å is usually a good compromise in most situations encoun‐ tered in affinity chromatography, since this gives fairly unrestricted diffusion for most

Particle diameters of the affinity supports are available in a wide variety. These range from HPLC-type materials with diameters of 10 µm or less to large particles for preparative work that have diameters of 400 µm. The purpose of the separation, mechanical properties of the support and the characteristics of the sample are important factors on the selection of particle size of the support. From a theoretical viewpoint, it is always advantageous to have a small particle size, since this will promote fast mass transfer of a solute between the outer flow stream and interior of a support particle. Sample molecules are transported down through the column by the flow of the mobile phase in the spaces between the support particles. To reach the affinity ligands, these molecules must diffuse through the stagnant mobile-phase layer surrounding the particles (i.e., the film model) and proceed to the inside pore network (Figure 6). It is where the sample molecules will finally bind to the affinity ligand. When the retained molecules are eluted, the same steps occur but in a reversed order. Smaller support particles mean shorter diffusion distances, since they have shorter pores and a thinner stagnant mobile phase layer

biomolecules while also providing a relatively large surface area for retention [8].

around and in the support. This results in shorter times needed for diffusion.

In preparative affinity chromatography, relatively large support particles are often used, making intraparticle diffusion the main factor limiting efficiency. In this case, diminishing the

**Figure 6.** Transport processes that occur in a chromatographic column.

solute to avoid severely restricted rates of diffusion.

Inorganic materials such as porous glass and silica are used when the extreme rigidity of the support material is needed [14]. Silica is especially used in the high-performance liquid affinity chromatography (HPLAC) or high-performance affinity chromatography (HPAC). Silicabased materials (Figure 5) are basically hydrophilic and they are suitable for affinity chroma‐ tography after they are modified at their surface. The native surface of silica is primarily covered with silanol groups which are weak acids and give strong negative charge to silica's surface at neutral pH. Irreversible adsorption of solutes (protein) can occur due to these charges and in combination with other binding forces. However, several methods can be used to render this surface inert toward such solutes, including polymer coating techniques and reactions between silica and alcohols or trialkoxysilanes [8]. They are also soluble at pH above 8 thus pH is an important parameter that limits the usage of silica [14].

**Figure 5. (a)** Structure of silica **(b)** SEM picture of typical silicagel [18]

A support material should be inert toward solutes. On the other hand easy coupling with ligand is also desired. Support materials are rich in hydroxyl groups, therefore attachment of ligands have been focused mainly on using these regions as anchoring points. Ideal affinity support should allow unhindered access of a solute to the immobilized ligand. For a macro‐ molecular solute, this requires a support that has large pores. Renkin equation can explain these pore sizes to be that allows one to estimate the effective diffusion coefficient (*D*eff) of a solute in a porous material.

$$D\_{\rm eff\ \bullet} D \quad K\_D \varepsilon\_p \text{f\u0} \\ 1-2.10 (R\_s/R\_p) + 2.09 (R\_s/R\_p)^3 - 0.095 (R\_s/R\_p)^5 \text{f\u0}$$

In this equation, *Rs/Rp* is the ratio of the solute's radius *(Rs)* to the pore radius *(Rp),* εp is the particle porosity, τ is the tortuosity factor, *KD* is the distribution coefficient for the solute, and *D* is the diffusion coefficient for the solute in free solution. By inserting different values for the ratio *Rs/Rp,* one finds that the pore diameter should be at least five times the diameter of the solute to avoid severely restricted rates of diffusion.

or have small pores. However this gel is resistant against enzymatic attacks and does not absorb

Inorganic materials such as porous glass and silica are used when the extreme rigidity of the support material is needed [14]. Silica is especially used in the high-performance liquid affinity chromatography (HPLAC) or high-performance affinity chromatography (HPAC). Silicabased materials (Figure 5) are basically hydrophilic and they are suitable for affinity chroma‐ tography after they are modified at their surface. The native surface of silica is primarily covered with silanol groups which are weak acids and give strong negative charge to silica's surface at neutral pH. Irreversible adsorption of solutes (protein) can occur due to these charges and in combination with other binding forces. However, several methods can be used to render this surface inert toward such solutes, including polymer coating techniques and reactions between silica and alcohols or trialkoxysilanes [8]. They are also soluble at pH above 8 thus

(a) (b)

A support material should be inert toward solutes. On the other hand easy coupling with ligand is also desired. Support materials are rich in hydroxyl groups, therefore attachment of ligands have been focused mainly on using these regions as anchoring points. Ideal affinity support should allow unhindered access of a solute to the immobilized ligand. For a macro‐ molecular solute, this requires a support that has large pores. Renkin equation can explain these pore sizes to be that allows one to estimate the effective diffusion coefficient (*D*eff) of a

In this equation, *Rs/Rp* is the ratio of the solute's radius *(Rs)* to the pore radius *(Rp),* εp is the particle porosity, τ is the tortuosity factor, *KD* is the distribution coefficient for the solute, and *D* is the diffusion coefficient for the solute in free solution. By inserting different values for the

biomolecules, thus it is used widely despite its mechanic disadvantages [8,14].

pH is an important parameter that limits the usage of silica [14].

O O

**Figure 5. (a)** Structure of silica **(b)** SEM picture of typical silicagel [18]

*Deff* <sup>=</sup>*D KDε<sup>p</sup>* 1−2.10(*Rs* / *Rp*) + 2.09(*Rs* / *Rp*)<sup>3</sup> −0.095(*Rs* / *Rp*)<sup>5</sup> / *t*

Si

Si O Si O Si

OH OH OH

O

66 Column Chromatography

Si Si

solute in a porous material.

For a protein in normal size (i.e., a diameter around 60 Å), a ratio of five for *Rp/Rs* means that the support pores should be in the range of 300 Å. Several common supports are available with such pore sizes. Support materials with very large pores give essentially unhindered diffusion for most solutes, but they also have a smaller surface area per milliliter of bed volume than supports with smaller pores. This reduced surface area leads to a diminished binding capacity. As a rule, a pore size of 300 to 700 Å is usually a good compromise in most situations encoun‐ tered in affinity chromatography, since this gives fairly unrestricted diffusion for most biomolecules while also providing a relatively large surface area for retention [8].

Particle diameters of the affinity supports are available in a wide variety. These range from HPLC-type materials with diameters of 10 µm or less to large particles for preparative work that have diameters of 400 µm. The purpose of the separation, mechanical properties of the support and the characteristics of the sample are important factors on the selection of particle size of the support. From a theoretical viewpoint, it is always advantageous to have a small particle size, since this will promote fast mass transfer of a solute between the outer flow stream and interior of a support particle. Sample molecules are transported down through the column by the flow of the mobile phase in the spaces between the support particles. To reach the affinity ligands, these molecules must diffuse through the stagnant mobile-phase layer surrounding the particles (i.e., the film model) and proceed to the inside pore network (Figure 6). It is where the sample molecules will finally bind to the affinity ligand. When the retained molecules are eluted, the same steps occur but in a reversed order. Smaller support particles mean shorter diffusion distances, since they have shorter pores and a thinner stagnant mobile phase layer around and in the support. This results in shorter times needed for diffusion.

**Figure 6.** Transport processes that occur in a chromatographic column.

In preparative affinity chromatography, relatively large support particles are often used, making intraparticle diffusion the main factor limiting efficiency. In this case, diminishing the particle size will increase the rate of movement of solutes between the support and surround‐ ing flow stream, giving an improved column performance. It is this effect that was the original driving force behind the use of smaller supports in affinity columns, thus giving rise to the technique of HPLAC. Under such conditions, a decrease in particle size by a factor of five can make it possible to increase the flow rate by up to 25-fold and still retain good chromatographic performance. This results in a dramatic improvement in the productivity of the system. However, a point is eventually reached when a decrease in particle diameter no longer gives a proportional improvement in an affinity column's performance. This has been observed in many analytical-scale systems that use HPLC-type supports with particle sizes less than 10 µm in diameter. Under these conditions, diffusion in the particle is now relatively fast, and it is the adsorption/desorption of sample molecules to and from the affinity ligand that becomes the limiting factor in speed and efficiency. Although better efficiency is always obtained with small support particles, using a small particle size tengenders some difficulties. One problem is the much higher flow resistance of these smaller particles. This increased flow resistance may lead to bed collapse when using soft gels such as agarose. And, although supports like silica can tolerate the higher pressures, that results, these will require the use of more expensive pumps to work at such pressure, as is generally done in HPLC. Another route that could be taken with small affinity supports is to use a short and wide column instead of a long and narrow one. The advantages of this are that the shorter, wider column can be run at higher flow rates without creating high-pressure drops. Another drawback with small particle sizes, especially in preparative work, is the increased danger of fouling that exists when particulate contaminants are in the feed stream or sample. This occurs because the interstitial spaces in a bed of small particles can be too narrow for such agents to pass through. Such fouling will increase the flow resistance and may lead to bed collapse if the support material does not have sufficient mechanical strength.

Membranes have been used for affinity chromatography in various formats, such as stacked sheets, in rolled geometries, or as hollow fibers. Materials that are commonly used for these membranes are cellulose, polysulfone, and polyamide. Because of their lack of diffusion pores, the surface area in these materials is as low as it is in nonporous beads. However, the flat geometry and shallow bed depth of membranes keep the pressure drop across them to a minimum degree. This means that high flow rates can be used, which makes these membranes

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As stated earlier, porous supports with a larger diameter facilitate low column backpressures and allow easy passage of contaminants through the column. But it is also necessary to keep the diameter of these supports as small as possible to diminish diffusion distances and thereby improve their chromatographic performance. One solution of these contradictory require‐ ments is to use particles that allow the flow of mobile phase directly through some of the pores. This is done in materials known as *perfusion media* or *through-pore particles*. Flow-through particles were initially developed in the early 1990s for ion-exchange chromatography and

Flow-through particles have a bimodal pore configuration, in which both small diffusion pores and large flow-through pores are present. Substances applied to a bed of this support are transported by mobile phase flow to the interior of each particle, leaving only short distances to be covered by diffusion to the support's surface (Figure 7). This combination leads to a dramatic improvement in performance compared with standard porous particles of the same size. This improvement is most pronounced in situations where slow diffusion is a limiting factor, such as in the chromatography of large molecules (e.g., proteins) at high flow rates.

**Figure 7.** Comparison of a particle with normal porosity versus a particle that contains flow-through pores. The nor‐

mal particle has long diffusion distances, whereas the flow-through particle has short diffusion distances.

especially well-suited for capturing proteins from dilute feed streams.

were later adapted for use in affinity chromatography.

As a result of these various requirements, the particle size to pick when designing a new affinity adsorbent will be a compromise between the desired chromatographic performances, prop‐ erties of the feed stream, and the mechanical strength of the support. Some common selections made in specific cases will be described in the next few sections.

Porous supports like agarose, polymethacrylate, or silica beads are generally used in current applications of affinity chromatography. However, in the past several years other types of supports have also become available commercially. Many of these newer materials have properties that give them superior performance in certain applications. Materials that fall in this category include; nonporous supports, membranes, flow-through beads, continuous beds and expanded-bed particles.

Nonporous beads with diameters of 1 to 3 µm can be an optimum choice for fast analytical or micropreparative separations, since the limiting factor of pore diffusion is virtually eliminated in these materials. Such beads may also be the best choice for fundamental or quantitative studies of affinity interactions, since the binding and dissociation behavior observed in these materials should be more directly linked with the interactions occurring between solutes and the affinity ligand. However, there is a substantial loss of surface area and binding capacity.

Membranes have been used for affinity chromatography in various formats, such as stacked sheets, in rolled geometries, or as hollow fibers. Materials that are commonly used for these membranes are cellulose, polysulfone, and polyamide. Because of their lack of diffusion pores, the surface area in these materials is as low as it is in nonporous beads. However, the flat geometry and shallow bed depth of membranes keep the pressure drop across them to a minimum degree. This means that high flow rates can be used, which makes these membranes especially well-suited for capturing proteins from dilute feed streams.

particle size will increase the rate of movement of solutes between the support and surround‐ ing flow stream, giving an improved column performance. It is this effect that was the original driving force behind the use of smaller supports in affinity columns, thus giving rise to the technique of HPLAC. Under such conditions, a decrease in particle size by a factor of five can make it possible to increase the flow rate by up to 25-fold and still retain good chromatographic performance. This results in a dramatic improvement in the productivity of the system. However, a point is eventually reached when a decrease in particle diameter no longer gives a proportional improvement in an affinity column's performance. This has been observed in many analytical-scale systems that use HPLC-type supports with particle sizes less than 10 µm in diameter. Under these conditions, diffusion in the particle is now relatively fast, and it is the adsorption/desorption of sample molecules to and from the affinity ligand that becomes the limiting factor in speed and efficiency. Although better efficiency is always obtained with small support particles, using a small particle size tengenders some difficulties. One problem is the much higher flow resistance of these smaller particles. This increased flow resistance may lead to bed collapse when using soft gels such as agarose. And, although supports like silica can tolerate the higher pressures, that results, these will require the use of more expensive pumps to work at such pressure, as is generally done in HPLC. Another route that could be taken with small affinity supports is to use a short and wide column instead of a long and narrow one. The advantages of this are that the shorter, wider column can be run at higher flow rates without creating high-pressure drops. Another drawback with small particle sizes, especially in preparative work, is the increased danger of fouling that exists when particulate contaminants are in the feed stream or sample. This occurs because the interstitial spaces in a bed of small particles can be too narrow for such agents to pass through. Such fouling will increase the flow resistance and may lead to bed collapse if the support material does not have

As a result of these various requirements, the particle size to pick when designing a new affinity adsorbent will be a compromise between the desired chromatographic performances, prop‐ erties of the feed stream, and the mechanical strength of the support. Some common selections

Porous supports like agarose, polymethacrylate, or silica beads are generally used in current applications of affinity chromatography. However, in the past several years other types of supports have also become available commercially. Many of these newer materials have properties that give them superior performance in certain applications. Materials that fall in this category include; nonporous supports, membranes, flow-through beads, continuous beds

Nonporous beads with diameters of 1 to 3 µm can be an optimum choice for fast analytical or micropreparative separations, since the limiting factor of pore diffusion is virtually eliminated in these materials. Such beads may also be the best choice for fundamental or quantitative studies of affinity interactions, since the binding and dissociation behavior observed in these materials should be more directly linked with the interactions occurring between solutes and the affinity ligand. However, there is a substantial loss of surface area and binding capacity.

made in specific cases will be described in the next few sections.

sufficient mechanical strength.

68 Column Chromatography

and expanded-bed particles.

As stated earlier, porous supports with a larger diameter facilitate low column backpressures and allow easy passage of contaminants through the column. But it is also necessary to keep the diameter of these supports as small as possible to diminish diffusion distances and thereby improve their chromatographic performance. One solution of these contradictory require‐ ments is to use particles that allow the flow of mobile phase directly through some of the pores. This is done in materials known as *perfusion media* or *through-pore particles*. Flow-through particles were initially developed in the early 1990s for ion-exchange chromatography and were later adapted for use in affinity chromatography.

Flow-through particles have a bimodal pore configuration, in which both small diffusion pores and large flow-through pores are present. Substances applied to a bed of this support are transported by mobile phase flow to the interior of each particle, leaving only short distances to be covered by diffusion to the support's surface (Figure 7). This combination leads to a dramatic improvement in performance compared with standard porous particles of the same size. This improvement is most pronounced in situations where slow diffusion is a limiting factor, such as in the chromatography of large molecules (e.g., proteins) at high flow rates.

**Figure 7.** Comparison of a particle with normal porosity versus a particle that contains flow-through pores. The nor‐ mal particle has long diffusion distances, whereas the flow-through particle has short diffusion distances.

Another format developed in 1990s was the *continuous bed* or *monolithic support*. Continuous bed supports consist of a single piece of material intersected by pores large enough to support chromatographic flow through the bed. Continuous beds have been developed using many well-known chromatographic materials, such as polyacrylamide, silica, polystyrene/polyme‐ thacrylates, cellulose, and agarose. Most of these continuous beds have two types of pores: large flow-carrying pores and smaller diffusion pores.

of existing molecular structures (triaznyl nucleotide-mimetics, purine and pirimidine deriva‐ tives, non-natural peptides, triazinyl dyes, other triazine-based ligands, oligosaccharide and boronic acid analogues). These can be generated by rational design or selected from ligand

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**•** The rational method features the functional approach and structural template approach.

**•** The combinatorial method relies on the selection of ligands from a library of synthetic

**•** The combined method employs both methods the ligand is selected from an intentionally

Many parameters have to be taken into account in order to select appropriate ligand. Table 1 exhibits the advantages and disadvantages of synthetic and biological ligands. Selectivity and affinity are the main advantages of biological ligands. Such ligands can be generated by *in vitro* evolution approaches and selecting from large combinatorial ligand libraries based on biological/genetic packages. Protein ligands display special advantages for example; higher affinities, higher proteolytic stability, preservation of their biological activity when produced by fusion to a different protein or domain. However these ligands can be expensive and unstable to the sterilization and cleanin conditions used in manufacturing process of biologics because of their biological origin, chemical nature and production methods. There is high contamination risk of the end-product with potentially dangerous leaches, in addition to

**Synthetic ligands Biological ligands**

Despite the advantages of the affinity chromatography technique, its use is limited due to high cost of affinity ligands and their biological and chemical instability. The develop‐ ment of methods for production of stable synthetic ligands has enabled "utilization of these materials in large scale. For the design of synthetic ligands, information about structure of the target protein and a potential binding site are required, thus a structure-based design can be achieved, in case correct prediction of the ligand's comformation and the binding affinity of the designed ligand. Function-based design can be applied when the structure of the target is not known [9]. Substantially, selection and design of ligands may be performed by using a template which is a part of a natural protein-ligand couple, model‐

libraries. Synthetic ligands are generated using three methods;

prepared library based on a rationally designed ligand.

possible contaminants originated from the biological source [20].

Capacity High Low to medium Cost Low to medium Medium to high Selectivity Medium to high Very high Stability High Low to medium

Toxicity Medium Low

**Table 1.** Comparison of biological and synthetic ligands [20]

ligands synthesized randomly.

The preparation of a continuous bed is usually straightforward. These beds can often be prepared directly in a chromatographic column, thereby avoiding the time-consuming steps of size classification and column packing that are normally needed with particle-based supports. Reports using continuous beds in affinity chromatography have shown that the efficiency of these materials is as good as that for particle-based supports.

In order to avoid column clogging, various pretreatment methods like filtration and centrifu‐ gation are often necessary to remove particulate matter from samples. To cut down on the need for such methods, a new class of adsorbents has recently been developed to handle viscous and particle-containing feed streams. These materials are known as *expanded-bed adsorbents*. In expanded-bed chromatography the direction of mobile phase flow is upward through the column and is fast enough to fluidize the support particles in the column. This causes the column bed to expand. This expansion makes the interstitial spaces in the column bed larger so that solid contaminants like cells and cell debris can pass through, thereby avoiding column clogging.

Another new type of expanded-bed adsorbent uses a thin layer of active material (i.e., derivatized agarose) that surrounds a heavy core. These adsorbents have small diffusion distances for biomolecules along with a higher density than other expanded-bed particles. The advantage of this combination is that it allows better chromatographic efficiencies to be obtained at higher flow rates [8].
