**1. Introduction**

Ion-exchange chromatography (IEC) is part of ion chromatography which is an important analytical technique for the separation and determination of ionic compounds, together with ion-partition/interaction and ion-exclusion chromatography [1]. Ion chromatography separa‐ tion is based on ionic (or electrostatic) interactions between ionic and polar analytes, ions present in the eluent and ionic functional groups fixed to the chromatographic support. Two distinct mechanisms as follows; ion exchange due to competitive ionic binding (attraction) and ion exclusion due to repulsion between similarly charged analyte ions and the ions fixed on the chromatographic support, play a role in the separation in ion chromatography. Ion exchange has been the predominant form of ion chromatography to date [2]. This chromatog‐ raphy is one of the most important adsorption techniques used in the separation of peptides, proteins, nucleic acids and related biopolymers which are charged molecules in different molecular sizes and molecular nature [3-6]. The separation is based on the formation of ionic bonds between the charged groups of biomolecules and an ion-exchange gel/support carrying the opposite charge [7]. Biomolecules display different degrees of interaction with charged chromatography media due to their varying charge properties [8].

The earliest report of ion-exchange chromatography date back to 1850, Thompson studied the adsorption of ammonium ions to soils [9-11]. Spedding and Powell published a series of papers describing practical methods for preparative separation of the rare earths by displacement ionexchange chromatography in 1947. Beginning in the 1950s, Kraus and Nelson reported numerous analytical methods which are used for metal ions based on separation of their chloride, fluoride, nitrate or sulfate complexes by anion chromatography [12]. In order to separate proteins an ion exchange chromatographic method was reported by Peterson and Sober in 1956. In modern form ion exchange chromatography was introduced by Small, Stevens and Bauman in 1975 [3]. Gjerde et al. published a method for anion chromatography in 1979 and this was followed by a similar method for cation chromatography in 1980 [12]. Ion-

© 2013 Acikara; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Acikara; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

exchange chromatography has been used for many years to separate various ionic compounds; cations and anions and still continues to be used. The popularity of ion exchange chromatog‐ raphy has been increased in recent years because this technique allows analysis of wide range of molecules in pharmaceutical, biotechnology, environmental, agricultural and other industries [2].

### **1.1. Ion exchange mechanism**

Ion-exchange chromatography which is designed specifically for the separation of differently charged or ionizable compounds comprises from mobile and stationary phases similar to other forms of column based liquid chromatography techniques [9-11]. Mobil phases consist an aqueous buffer system into which the mixture to be resolved. The stationary phase usually made from inert organic matrix chemically derivative with ionizable functional groups (fixed ions) which carry displaceable oppositely charged ion [11]. Ions which exist in a state of equilibrium between the mobile phase and stationary phases giving rise to two possible formats, anion and cation exchange are referred to as counter ion (Figure 1) [1,13]. Exchange‐ able matrix counter ions may include protons (H+ ), hydroxide groups (OH- ), single charged mono atomic ions (Na+ , K+ , Cl- ), double charged mono atomic ions (Ca2+, Mg2+), and polyatomic inorganic ions (SO4 2-, PO4 3-) as well as organic bases (NR2H+ ) and acids (COO- ) [11]. Cations are separated on cation-exchange resin column and anions on an anion exchange resin column [10]. Separation based on the binding of analytes to positively or negatively charged groups which are fixed on a stationary phase and which are in equilibrium with free counter ions in the mobile phase according to differences in their net surface charge (Figure 1) [13-14].

S-X-

S-X<sup>+</sup>

ion X+

anion X-

C<sup>+</sup> + M+↔ S-X-

represented as follow;

A- + B-

In this process the cation M+

↔ S-X<sup>+</sup>

B- + A-

resulting in the separation due to ion-exchange [2].

salt concentration in the mobile phase [11].

M<sup>+</sup> + C<sup>+</sup>

of the eluent replaced with the analyte cation C+ bound to the

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33

which is fixed on the surface of the chromatographic support (S).

In anion exchange chromatography, the exchanging ions are anions and the equation is

The anion B- of the eluent replaced with the analyte cation A- bound to the positively charged

Molecules vary considerably in their charge properties and will exhibit different degrees of interaction with charged chromatography support according to differences in their overall charge, charge density and surface charge distribution. Net surface charge of all molecules with ionizable groups is highly pH dependent [13]. Therefore pH of the mobile phase should be selected according to the net charge on a protein of interest within a mixture is opposite to that of matrix functional group, that it will displace the functional group counter ion and bind the matrix. On the other hand oppositely charged proteins will not be retained. Adsorbed protein analytes can be eluted by changing the mobile phase pH which effect the net charge of adsorbed protein, so its matrix binding capacity. Moreover increasing the concentration of a similarly charged species within the mobile phase can be resulted in elution of bound proteins. During ion exchange chromatography for example in anion exchange as illustrated in Figure 2, negatively charged protein analytes can be competitively displaced by the addition of negatively charged ions. The affinity of interaction between the salt ions and the functional groups will eventually exceed that the interaction exists between the protein charges and the functional groups, resulting in protein displacement and elution by increasing gradually the

Complex mixtures of anions or cations can usually be separated and quantitative amounts of each ion measured in a relatively short time by ion exchange chromatography [10]. In classical ion-exchange chromatography separations have been performed in the open-column mode. Column which is loosely packed with stationary phase as small particles made of 1-2 cm diameter glass. The mobile phase or eluent contains the competing ion and is passed contin‐ uously into the column and percolates through it under gravity. Sample mixture is applied to the top of the column and allowed to pass into the bed of ion- exchange material. Eluent flow is then resumed and fractions of eluent are collected at regular intervals from the column outlet. Open column ion-exchange chromatography is very slow due to low eluent flow-rates. Increasing flow rate may result in deteriorated separation efficiency (Figure 3). In modern ionexchange chromatography the usage of high efficiency ion exchange materials combined with flow-through detection have overcome of these challenges. Separations are performed on the column which is filled with ion-exchanger as particles in uniform size. The particles of ionexchange material are generally very much smaller than those used for classical open column

 on the surface of the stationary phase. The adsorption of the analyte to the stationary phase and desorption by the eluent ions is repeated during their journey in the column,

### **Figure 1.** Types of ion exchangers

Ion exchange chromatography involves separation of ionic and polar analytes using chromatographic supports derivatized with ionic functional groups that have charges opposite that of the analyte ions. The analyte ions and similarly charged ions of the eluent compete to bind to the oppositely charged ionic functional group on the surface of the stationary phase. Assuming that the exchanging ions (analytes and ions in the mobile phase) are cations, the competition can be explained using the following equation;

S-X-C<sup>+</sup> + M+↔ S-X-M<sup>+</sup> + C<sup>+</sup>

exchange chromatography has been used for many years to separate various ionic compounds; cations and anions and still continues to be used. The popularity of ion exchange chromatog‐ raphy has been increased in recent years because this technique allows analysis of wide range of molecules in pharmaceutical, biotechnology, environmental, agricultural and other

Ion-exchange chromatography which is designed specifically for the separation of differently charged or ionizable compounds comprises from mobile and stationary phases similar to other forms of column based liquid chromatography techniques [9-11]. Mobil phases consist an aqueous buffer system into which the mixture to be resolved. The stationary phase usually made from inert organic matrix chemically derivative with ionizable functional groups (fixed ions) which carry displaceable oppositely charged ion [11]. Ions which exist in a state of equilibrium between the mobile phase and stationary phases giving rise to two possible formats, anion and cation exchange are referred to as counter ion (Figure 1) [1,13]. Exchange‐

3-) as well as organic bases (NR2H+

are separated on cation-exchange resin column and anions on an anion exchange resin column [10]. Separation based on the binding of analytes to positively or negatively charged groups which are fixed on a stationary phase and which are in equilibrium with free counter ions in the mobile phase according to differences in their net surface charge (Figure 1) [13-14].


anion exchanger stationary phase particle cation exchanger stationary phase particle

Ion exchange chromatography involves separation of ionic and polar analytes using chromatographic supports derivatized with ionic functional groups that have charges opposite that of the analyte ions. The analyte ions and similarly charged ions of the eluent compete to bind to the oppositely charged ionic functional group on the surface of the stationary phase. Assuming that the exchanging ions (analytes and ions in the mobile

phase) are cations, the competition can be explained using the following equation;

), hydroxide groups (OH-

\_ \_ \_ \_ \_ \_ \_ \_ \_ \_

) and acids (COO-

Positively charged analyte (cation)

), double charged mono atomic ions (Ca2+, Mg2+), and polyatomic

), single charged

) [11]. Cations

industries [2].

32 Column Chromatography

**1.1. Ion exchange mechanism**

mono atomic ions (Na+

inorganic ions (SO4

able matrix counter ions may include protons (H+

, K+ , Cl-

2-, PO4

Negatively charged analyte (anion)

+ + + + + + + + + + +

**Figure 1.** Types of ion exchangers

In this process the cation M+ of the eluent replaced with the analyte cation C+ bound to the anion X which is fixed on the surface of the chromatographic support (S).

In anion exchange chromatography, the exchanging ions are anions and the equation is represented as follow;

S-X<sup>+</sup> A- + B- ↔ S-X<sup>+</sup> B- + A-

The anion B- of the eluent replaced with the analyte cation A- bound to the positively charged ion X+ on the surface of the stationary phase. The adsorption of the analyte to the stationary phase and desorption by the eluent ions is repeated during their journey in the column, resulting in the separation due to ion-exchange [2].

Molecules vary considerably in their charge properties and will exhibit different degrees of interaction with charged chromatography support according to differences in their overall charge, charge density and surface charge distribution. Net surface charge of all molecules with ionizable groups is highly pH dependent [13]. Therefore pH of the mobile phase should be selected according to the net charge on a protein of interest within a mixture is opposite to that of matrix functional group, that it will displace the functional group counter ion and bind the matrix. On the other hand oppositely charged proteins will not be retained. Adsorbed protein analytes can be eluted by changing the mobile phase pH which effect the net charge of adsorbed protein, so its matrix binding capacity. Moreover increasing the concentration of a similarly charged species within the mobile phase can be resulted in elution of bound proteins. During ion exchange chromatography for example in anion exchange as illustrated in Figure 2, negatively charged protein analytes can be competitively displaced by the addition of negatively charged ions. The affinity of interaction between the salt ions and the functional groups will eventually exceed that the interaction exists between the protein charges and the functional groups, resulting in protein displacement and elution by increasing gradually the salt concentration in the mobile phase [11].

Complex mixtures of anions or cations can usually be separated and quantitative amounts of each ion measured in a relatively short time by ion exchange chromatography [10]. In classical ion-exchange chromatography separations have been performed in the open-column mode. Column which is loosely packed with stationary phase as small particles made of 1-2 cm diameter glass. The mobile phase or eluent contains the competing ion and is passed contin‐ uously into the column and percolates through it under gravity. Sample mixture is applied to the top of the column and allowed to pass into the bed of ion- exchange material. Eluent flow is then resumed and fractions of eluent are collected at regular intervals from the column outlet. Open column ion-exchange chromatography is very slow due to low eluent flow-rates. Increasing flow rate may result in deteriorated separation efficiency (Figure 3). In modern ionexchange chromatography the usage of high efficiency ion exchange materials combined with flow-through detection have overcome of these challenges. Separations are performed on the column which is filled with ion-exchanger as particles in uniform size. The particles of ionexchange material are generally very much smaller than those used for classical open column

ion-exchange chromatography [1]. However ion-exchange resins used in modern chromatog‐ raphy have lower capacity than older resins [10]. The eluent must be pumped through the column due to the small particle size of stationary phase. The sample mixture is applied into eluent by the injection port. Finally the separated ions are detected with a flow-through

> Lehninger Principles of Biochemistry, Fifth Edition, © 2008 W.H. Freeman and

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35

Company

This technique has been used for the analyses of anions and cations, including metal ions, mono- and oligosaccharides, alditols and other polyhydroxy compounds, aminoglycosides (antibiotics), amino acids and peptides, organic acids, amines, alcohols, phenols, thiols, nucleotides and nucleosides and other polar molecules. It has been successfully applied to the analysis of raw materials, bulk active ingredients, counter ions, impurities, and degradation products, excipients, diluents and at different stages of the production process as well as for the analysis of production equipment cleaning solutions, waste streams, container compati‐ bility and other applications [2]. Wide applicability including high performance and highthroughput application formats, average cost, powerful resolving ability, large sample handling capacity and ease of scale-up as well as automation allow the ion exchange chroma‐ tography has become one of the most important and extensively used of all liquid chromato‐

Although the extensive use of ion exchange chromatography the mechanism of the separation has not completely been elucidated. A considerable effort has been made to describe the ion exchange process theoretically [3,9]. One of the important disadvantages of this technique is

detection instrument [1].

Protein mixture

Polymer beads with negatively charged functional

graphic technique [11].

**Figure 3.** Ion exchange chromatography technique

groups

**Figure 2.** Separation steps in anion exchange chromatography (GE Healthcare)

ion-exchange chromatography [1]. However ion-exchange resins used in modern chromatog‐ raphy have lower capacity than older resins [10]. The eluent must be pumped through the column due to the small particle size of stationary phase. The sample mixture is applied into eluent by the injection port. Finally the separated ions are detected with a flow-through detection instrument [1].

**Figure 3.** Ion exchange chromatography technique

matrix

neutral or positively charged proteins

> negatively charged proteins

fixed ionic groups on matrix **Equilibration**

starting buffer

**Sample application**

application

**Elution**

**Elution**

**Elution**

**Washing**

equilibration

ion exchange medium equilibrated with

Oppositely charged proteins bind to ionic groups or to the ion exchange medium, becoming concentrated on the column. Uncharged proteins or those with the same charge as the ionic groups are eluted during or just after sample

Increasing ionic strength (using a gradient) displaces bound proteins as ions in the buffer compete for binding sites

Further increases in ionic strength displace proteins that are more highly charged

Further increases in ionic strength displace proteins that are more highly charged

Final high ionic strength wash removes any ionically bound proteins before re-

buffer ionic groups

34 Column Chromatography

**Figure 2.** Separation steps in anion exchange chromatography (GE Healthcare)

This technique has been used for the analyses of anions and cations, including metal ions, mono- and oligosaccharides, alditols and other polyhydroxy compounds, aminoglycosides (antibiotics), amino acids and peptides, organic acids, amines, alcohols, phenols, thiols, nucleotides and nucleosides and other polar molecules. It has been successfully applied to the analysis of raw materials, bulk active ingredients, counter ions, impurities, and degradation products, excipients, diluents and at different stages of the production process as well as for the analysis of production equipment cleaning solutions, waste streams, container compati‐ bility and other applications [2]. Wide applicability including high performance and highthroughput application formats, average cost, powerful resolving ability, large sample handling capacity and ease of scale-up as well as automation allow the ion exchange chroma‐ tography has become one of the most important and extensively used of all liquid chromato‐ graphic technique [11].

Although the extensive use of ion exchange chromatography the mechanism of the separation has not completely been elucidated. A considerable effort has been made to describe the ion exchange process theoretically [3,9]. One of the important disadvantages of this technique is that this method provides no direct information on events occurring at the surface of the stationary phase, because the ion-exchange equilibrium is always determined by the balance between the solute interaction and the eluent interaction with the active sites of resin [3]. Ion exchange is similar to sorption, since in both cases a solid takes up dissolved sample. The most important difference between them is in stoichiometric nature of ion exchange. Each ion removed from the solution is replaced by an equivalent amount of another ion of the same charge, while a solute is usually taken-up non-stoichiometrically without being replaced in sorption [15]. Stoichiometric displacement based on the mass action law and describes the retention of a solute ion as an exchange process with the counter ion bound to the surface [9]. According to this model, the retention of a protein under isocratic, linear conditions is related to counter ion concentration and can be represented by equilibrium as follow;

The value of DA is dependent on the size of the population of molecules of component A in the stationary and eluent phases [1]. As the equilibrium is dynamic, there is a continual, rapid interchange of molecules of component A between the two phases. The fraction of time, fm,

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The mechanism of the anion and cation exchange are very similar. When analytes enter to the ion exchange column, firstly they bind to the oppositely charged ionic sites on the stationary phase through the Coulombic attraction [2]. In accordance with Coulomb's law, the interac‐ tions between ions in the solute and oppositely charged ligands on the matrix in ion-exchange chromatography are due to the electrostatic forces. Coulomb's law is given by the equation as

If the charges on both ions are same (both are positive or negative) the force is repulsive, if they are different (one positive and the other negative) the force is attractive. When the ion charge of the species increase (Divalent ion should interact more strongly than a monovalent ion) and when the dielectric constant decrease (Two oppositely charged molecules increased more strongly in an organic solvent than in water), the interactions increase. On the other hand the distance between the charges increases the interactions decrease. Additionally, other interactions, especially, van der Waals forces participate to the Coulombic forces [2,17].

Ion exchange chromatography, which is also known as adsorption chromatography, is a useful

that an average molecule of A spends in the mobile phase is given by:

fm = Amount of A in the mobile phase / Total amount of A

fm = [A]m Vm / [A]m Vm + [A]r w

w: Weight of the stationary phase

Vm: Volume of the mobile phase [1]

f: Interaction electrostatic force

: Dielectric constant of the medium

r: The distance between charges.

and popular method due to its;

**•** mild separation conditions,

**•** high capacity,

**•** high resolving power,

q1q2: The charge on ions

= 1/ 1+ DA (w / Vm)

k' = DA (W / Vm)

fm = 1 / 1 + k'

follow;

f=q1q2/εr<sup>2</sup>

log *k* = -(Zp/Zs) log *C*<sup>m</sup> + log(*φQ*)

*k* is the retention factor and Cm is the concentration of the counter ion in the mobile phase. *Z*p/ *Z*s (= *Z*) is the ratio of the characteristic charge of the protein to the value of the counter ion and presents a statistical average of the electrostatic interactions of the protein with the stationary phase as it migrates through the column. The behavior of ion exchange chromatographic system can be explained by stoichiometric models. However, the mechanism of the ion exchange separation is more complex and stiochiometric consideration is inapplicable to longrange mechanisms, such as electrostatic interactions due to the distribution of ions in solution is also influenced by the electrostatic potential [3,6]. Other interactions between solute-solute, solute-solvent and solvent-solvent also contribute to retention and selectivity in ion exchange. For example ion-dipole and dispersion interaction, should be included as important mecha‐ nisms. Additionally entropic contribution originating from solvent, such as water, structures around ion exchange sites should also be regarded as important [3]. In addition to these the primary separation mechanism is the electrostatic interaction between ion-exchange sites and counter ions in ion exchange chromatography [6].

An important feature differentiating the ion exchange resins from other types of gels is the presence of functional groups. The groups are attached to the matrix. The ion exchange process between the ions in the solution takes place on these functional groups. The exchange of ions between the ion exchange resin and the solution is governed by two principles:


Equilibrium is established for each sample component between the eluent and stationary phases when a sample is introduced into the ion-exchange chromatography. The distribution of component (A) between the two phases is expressed by the distribution coefficient, "DA".

DA= A r/ A <sup>m</sup>

The value of DA is dependent on the size of the population of molecules of component A in the stationary and eluent phases [1]. As the equilibrium is dynamic, there is a continual, rapid interchange of molecules of component A between the two phases. The fraction of time, fm, that an average molecule of A spends in the mobile phase is given by:

fm = Amount of A in the mobile phase / Total amount of A

fm = [A]m Vm / [A]m Vm + [A]r w = 1/ 1+ DA (w / Vm) k' = DA (W / Vm) fm = 1 / 1 + k'

w: Weight of the stationary phase

Vm: Volume of the mobile phase [1]

The mechanism of the anion and cation exchange are very similar. When analytes enter to the ion exchange column, firstly they bind to the oppositely charged ionic sites on the stationary phase through the Coulombic attraction [2]. In accordance with Coulomb's law, the interac‐ tions between ions in the solute and oppositely charged ligands on the matrix in ion-exchange chromatography are due to the electrostatic forces. Coulomb's law is given by the equation as follow;

f=q1q2/εr<sup>2</sup>

that this method provides no direct information on events occurring at the surface of the stationary phase, because the ion-exchange equilibrium is always determined by the balance between the solute interaction and the eluent interaction with the active sites of resin [3]. Ion exchange is similar to sorption, since in both cases a solid takes up dissolved sample. The most important difference between them is in stoichiometric nature of ion exchange. Each ion removed from the solution is replaced by an equivalent amount of another ion of the same charge, while a solute is usually taken-up non-stoichiometrically without being replaced in sorption [15]. Stoichiometric displacement based on the mass action law and describes the retention of a solute ion as an exchange process with the counter ion bound to the surface [9]. According to this model, the retention of a protein under isocratic, linear conditions is related

*k* is the retention factor and Cm is the concentration of the counter ion in the mobile phase. *Z*p/ *Z*s (= *Z*) is the ratio of the characteristic charge of the protein to the value of the counter ion and presents a statistical average of the electrostatic interactions of the protein with the stationary phase as it migrates through the column. The behavior of ion exchange chromatographic system can be explained by stoichiometric models. However, the mechanism of the ion exchange separation is more complex and stiochiometric consideration is inapplicable to longrange mechanisms, such as electrostatic interactions due to the distribution of ions in solution is also influenced by the electrostatic potential [3,6]. Other interactions between solute-solute, solute-solvent and solvent-solvent also contribute to retention and selectivity in ion exchange. For example ion-dipole and dispersion interaction, should be included as important mecha‐ nisms. Additionally entropic contribution originating from solvent, such as water, structures around ion exchange sites should also be regarded as important [3]. In addition to these the primary separation mechanism is the electrostatic interaction between ion-exchange sites and

An important feature differentiating the ion exchange resins from other types of gels is the presence of functional groups. The groups are attached to the matrix. The ion exchange process between the ions in the solution takes place on these functional groups. The exchange of ions

**2.** The exchange reactions take place on the basis of equivalency in accordance with the principle of electro neutrality. The number of milimoles of an ion sorbed by an exchange should correspond to the number of milimoles of an equally charged ion that has been

Equilibrium is established for each sample component between the eluent and stationary phases when a sample is introduced into the ion-exchange chromatography. The distribution of component (A) between the two phases is expressed by the distribution coefficient, "DA".

between the ion exchange resin and the solution is governed by two principles:

**1.** The process is reversible, only rare exceptions are known

to counter ion concentration and can be represented by equilibrium as follow;

log *k* = -(Zp/Zs) log *C*<sup>m</sup> + log(*φQ*)

36 Column Chromatography

counter ions in ion exchange chromatography [6].

released from the ion exchange [16].

DA= A r/ A <sup>m</sup>

f: Interaction electrostatic force

q1q2: The charge on ions

: Dielectric constant of the medium

r: The distance between charges.

If the charges on both ions are same (both are positive or negative) the force is repulsive, if they are different (one positive and the other negative) the force is attractive. When the ion charge of the species increase (Divalent ion should interact more strongly than a monovalent ion) and when the dielectric constant decrease (Two oppositely charged molecules increased more strongly in an organic solvent than in water), the interactions increase. On the other hand the distance between the charges increases the interactions decrease. Additionally, other interactions, especially, van der Waals forces participate to the Coulombic forces [2,17].

Ion exchange chromatography, which is also known as adsorption chromatography, is a useful and popular method due to its;


General components of an ion-exchange chromatography are presented as below (Figure 4).


In ion-exchange chromatography, adsorption and desorption processes are determined by the properties of the three interacting entities;


### **1.2. Stationary phase**

Selection of a suitable ion-exchange matrix probably is the most important in ion exchange protocol and is based on various factors such as; ion exchanger charge/strength, linear flow rate/sample volume and sample properties [11]. In ion-exchange chromatography, numerous stationary phases are available from different manufacturers, which vary significantly in a number of chemical and physical properties [6,18]. Stationary phases comprised of two structural elements; the charged groups which are involved in the exchange process and the matrix on which the charged groups are fixed [18]. Ion exchangers are characterized both by the nature of the ionic species comprising the fixed ion and by the nature of the insoluble ionexchange matrix itself [1].

beads can be categorized as non-porous, microporous and macroporous. (Figure 5 and Figure 6) [14]. High porosity offers a large surface area covered by charged groups and so provides a high binding capacity [13]. However when compared with beaded matrix fibrous ion exchangers based on cellulose exhibit lower chromatographic resolution [14]. On the other hand high porosity is an advantage when separating large molecules [13] and prefractionation [14]. Non-porous matrices are preferable for high resolution separations when diffusion effects must be avoided [13]. Micropores increase the binding capacity but cause to a band broaden‐ ing. Another disadvantage of microporous beads is that protein can bind to the surface of the beads near to the pores, so penetration of proteins into the pores can prevent or slow down. These problems are overcome by using macroporous particles with pore diameters of about 600-800 nm which are introduced recently. These kinds of particles behave differently

 

Column

Detector

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Data processing

Thermostatted

Injection unit autosampler

Pump

pressure and flow indicator

 

**Figure 4.** Ion-exchange Chromatography System

Ion exchangers are called cation exchangers if they have negatively charged functional groups and possess exchangeable cations. Anion exchangers carry anions because of the positive charge of their fixed groups [15]. The charged groups determine the specifity and strength of protein binding by their polarity and density while the matrix determines the physical and chemical stability and the flow characteristics of the stationary phase and may be responsible for unspecific binding effects [18].

General structure (fibrous or beaded form), particle size and variation, pore structures and dimensions, surface chemistry (hydrophilic or hydrophobic), swelling characteristics of matrix are important factors which effect chromatographic resolution [11,18]. Porosity of ion exchange

**Figure 4.** Ion-exchange Chromatography System

 

**•** versatility and widespeared applicability,

properties of the three interacting entities;

**•** The constituents of the mobile phase

General components of an ion-exchange chromatography are presented as below (Figure 4).

In ion-exchange chromatography, adsorption and desorption processes are determined by the

Selection of a suitable ion-exchange matrix probably is the most important in ion exchange protocol and is based on various factors such as; ion exchanger charge/strength, linear flow rate/sample volume and sample properties [11]. In ion-exchange chromatography, numerous stationary phases are available from different manufacturers, which vary significantly in a number of chemical and physical properties [6,18]. Stationary phases comprised of two structural elements; the charged groups which are involved in the exchange process and the matrix on which the charged groups are fixed [18]. Ion exchangers are characterized both by the nature of the ionic species comprising the fixed ion and by the nature of the insoluble ion-

Ion exchangers are called cation exchangers if they have negatively charged functional groups and possess exchangeable cations. Anion exchangers carry anions because of the positive charge of their fixed groups [15]. The charged groups determine the specifity and strength of protein binding by their polarity and density while the matrix determines the physical and chemical stability and the flow characteristics of the stationary phase and may be responsible

General structure (fibrous or beaded form), particle size and variation, pore structures and dimensions, surface chemistry (hydrophilic or hydrophobic), swelling characteristics of matrix are important factors which effect chromatographic resolution [11,18]. Porosity of ion exchange

**•** A high pressure pump with pressure and flow indicator, to deliver the eluent

**•** A column, to separate the sample mixture into the individual components

**•** A data system for collecting and organizing the chromatograms and data

**•** A detector, to measure the analyte peaks as eluent from the column

**•** An injector for introducing the sample into the eluent stream and onto the column

**•** tendency to concentrate the sample

**•** relatively low cost [17].

38 Column Chromatography

**•** An oven, optional

**•** The stationary phase,

**•** The solute [18].

**1.2. Stationary phase**

exchange matrix itself [1].

for unspecific binding effects [18].

beads can be categorized as non-porous, microporous and macroporous. (Figure 5 and Figure 6) [14]. High porosity offers a large surface area covered by charged groups and so provides a high binding capacity [13]. However when compared with beaded matrix fibrous ion exchangers based on cellulose exhibit lower chromatographic resolution [14]. On the other hand high porosity is an advantage when separating large molecules [13] and prefractionation [14]. Non-porous matrices are preferable for high resolution separations when diffusion effects must be avoided [13]. Micropores increase the binding capacity but cause to a band broaden‐ ing. Another disadvantage of microporous beads is that protein can bind to the surface of the beads near to the pores, so penetration of proteins into the pores can prevent or slow down. These problems are overcome by using macroporous particles with pore diameters of about 600-800 nm which are introduced recently. These kinds of particles behave differently compared to microporous materials with respect to microflow characteristics the new term perfusion chromatography has been created [14].

**Figure 5.** Schematic presentation of different matrix types (a) non-porous beads (b) microporous beads (c) macropo‐ rous beads

**Figure 7.** Continuous bed matrix

for column cleaning [14].

Matrix materials;

In order to minimize non-specific interactions with sample components inert matrix should be used. High physical stability provides that the volume of the packed medium remains constant despite extreme changes in salt concentration or pH for improving reproducibility and avoiding the need to repack columns. High physical stability and uniformity of particle size facilitate high flow rates, particularly during cleaning or re-equilibration steps, to improve throughput and productivity [13]. There are pH and pressure limits for each stationary phases. For example pH values higher than 8 should not used in silica based materials which are not coated with organic materials. Matrix stability also should be considered when the chemicals such as organic solvents or oxidizing agents should be required to use or when they are chosen

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Matrices which are obtained by polymerization of polystyrene with varying amounts of divinylbenzene are known as the original matrices for ion exchange chromatography. However these matrices have very hydrophobic surface and proteins are irreversibly damaged due to strong binding. Ion exchangers which are based on cellulose with hydrophilic back‐ bones are more suitable matrices for protein separations. Other ion exchange matrices with

hydrophilic properties are based on agarose or dextran [14].

Several matrix types and their important properties can be listed as follow;

**•** Cellulose; Hydrophilic surface, enhanced stability by cross-linking, inexpensive

**Figure 6. (a)** Non-porous beads **(b)** Porous beads

Furthermore a new matrix type which has been recently introduced is based on a completely new principle and exhibits improved chromatographic features when compared with con‐ ventional ion exchangers. This matrix which is known as continuous bed does not consist of ion exchange beads or fibers. The matrix is synthesized in the column by polymerization and established from continuous porous support consisting of a nodule chains (Figure 7). The advantages of that matrix are mainly due to the more homogeneous mobile phase flow and short diffusion distances for the proteins. This is explained by the non-beaded form and the unique pore structure of the support [14].

Size, size distribution and porosity of the matrix particles are the main factors which affect the flow characteristics and chromatographic resolution. Small particles improved chromato‐ graphic resolution. Stationary phases with particle of uniform size are superior to heteroge‐ nous materials with respect to resolution and attainable flow rates. The pore size of ion exchange bead directly effect the binding capacity for a particular protein dependent on the molecular weight of the protein because it determines the access of proteins to the interior of the beads. Binding of large proteins can be restricted to the bead surface only so that the total binding capacity of the ion exchanger is not exploited Pore diameter of 30 nm is optimal for proteins up to a molecular weight of about 200.000 Da [14].

**Figure 7.** Continuous bed matrix

compared to microporous materials with respect to microflow characteristics the new term

(a) (b) (c)

**Figure 5.** Schematic presentation of different matrix types (a) non-porous beads (b) microporous beads (c) macropo‐

(a) (b)

Furthermore a new matrix type which has been recently introduced is based on a completely new principle and exhibits improved chromatographic features when compared with con‐ ventional ion exchangers. This matrix which is known as continuous bed does not consist of ion exchange beads or fibers. The matrix is synthesized in the column by polymerization and established from continuous porous support consisting of a nodule chains (Figure 7). The advantages of that matrix are mainly due to the more homogeneous mobile phase flow and short diffusion distances for the proteins. This is explained by the non-beaded form and the

Size, size distribution and porosity of the matrix particles are the main factors which affect the flow characteristics and chromatographic resolution. Small particles improved chromato‐ graphic resolution. Stationary phases with particle of uniform size are superior to heteroge‐ nous materials with respect to resolution and attainable flow rates. The pore size of ion exchange bead directly effect the binding capacity for a particular protein dependent on the molecular weight of the protein because it determines the access of proteins to the interior of the beads. Binding of large proteins can be restricted to the bead surface only so that the total binding capacity of the ion exchanger is not exploited Pore diameter of 30 nm is optimal for

perfusion chromatography has been created [14].

**Figure 6. (a)** Non-porous beads **(b)** Porous beads

unique pore structure of the support [14].

proteins up to a molecular weight of about 200.000 Da [14].

rous beads

40 Column Chromatography

In order to minimize non-specific interactions with sample components inert matrix should be used. High physical stability provides that the volume of the packed medium remains constant despite extreme changes in salt concentration or pH for improving reproducibility and avoiding the need to repack columns. High physical stability and uniformity of particle size facilitate high flow rates, particularly during cleaning or re-equilibration steps, to improve throughput and productivity [13]. There are pH and pressure limits for each stationary phases. For example pH values higher than 8 should not used in silica based materials which are not coated with organic materials. Matrix stability also should be considered when the chemicals such as organic solvents or oxidizing agents should be required to use or when they are chosen for column cleaning [14].

Matrices which are obtained by polymerization of polystyrene with varying amounts of divinylbenzene are known as the original matrices for ion exchange chromatography. However these matrices have very hydrophobic surface and proteins are irreversibly damaged due to strong binding. Ion exchangers which are based on cellulose with hydrophilic back‐ bones are more suitable matrices for protein separations. Other ion exchange matrices with hydrophilic properties are based on agarose or dextran [14].

Several matrix types and their important properties can be listed as follow;

Matrix materials;

**•** Cellulose; Hydrophilic surface, enhanced stability by cross-linking, inexpensive

**•** Dextran; Considerable swelling as a function of ionic milieu, improved materials by crosslinking)

tertiary and quaternary amines proved to be useful for ion exchange chromatography. Immobilized diethylaminoethyl and carboxymethyl groups are the most widely used ion

Theionexchangecapacityofanion-exchangerisdeterminedbythenumberoffunctionalgroups per unit weight of the resin [13]. The total ionic capacity is the number of charged functional groupspermlmedium, a fixedparameterof eachmediumandcanbegivenasmval/mlfor small ions. Density and accessibility of these charged groups both on the surface and within the pores define the total binding capacity. Ionic medium and the presence of other proteins if a particu‐ lar protein is considered also affect the binding capacity. However, under defined conditions, the amount of the certain protein which is bound to ion exchanger is more suitable parameter for determining and comparing the capacity of ion exchange chromatography. Albumin for anion exchangers and hemoglobin for cation exchangers is usually used for this purpose. Determinationofthebindingcapacitybeforetheexperimentisgenerallyrecommendedbecause the capacity for aparticularproteindepends on its size andalso on the sample composition.The binding capacity of a column can be increased for proteins which are retained on the column at high salt concentrations. The salt concentration is adjusted to a suitable concentration in which the protein of interest tightly bound to the ion exchanger while others which have lower affinity

**Buffer counter ions pH range Commercial samples**

Capto® S SP Sepharose® SP Sephadex® TSKgel SP\_5PW

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CM Cellulose CM Sepharose® CM Sephadex® CM Sepharose®

TSKgel CM-5PW

Q Sepharose® Capto® Q Dowex® 1X2

Amberlite®

Capto®

QAE Sephadex®

DEAE-Sepharose®

 DEAE DEAE Cellulose

CL6B

/ Amberjet®

Na+, H+, Li+ 4-13

, SO4 2- 2-12

, SO4 2- 2-9

pass through the column without occupying binding sites [14].

Weak cation Carboxylic acid Na+, H+, Li+ 6-10

Cl- , HCOO3 - ,

Cl- , HCOO3 - ,

CH3COO-

CH3COO-

**Exchange Type Ion exchange**

Strong anion Quaternary amine (Q)

Strong cation

Weak anion

**group**

Sulfonic acid (SP)

Primary amine Secondary amine Tertiary amine (DEAE)

**Table 1.** Weak and Strong type anion and cation exchangers

exchangers [11].


In addition to electrostatic interactions between stationary phase and proteins, some further mechanisms such as hydrophobic interactions, hydrogen bonding may contribute to protein binding. Hydrophobic interactions especially occur with synthetic resin ion exchangers such as which are produced by copolymerization of styrene and divinylbenzene. These materials are not usually used for separation of proteins. However new ion exchange materials that consist of styrene-divinylbenzene copolymer beads coated with hydrophilic ion exchanger film were introduced. According to the retention behavior of some proteins, it is considered that coating of the beads so efficient that unspecific binding due to hydrophobic interactions cannot be observed. Silica particles have also been coated with hydrophilic matrix. Acrylic acid polymers are also used for the protein separation in ion exchange chromatography. These polymers are especially suitable for purification of basic proteins [14].

The functional groups substituted onto a chromatographic matrix determine the charge of an ion exchange medium; positively-charged anion exchanger or a negatively-charged cation exchanger [13]. Both exchangers can be further classified as strong and weak type as shown in Table 1. The terms weak and strong are not related to the binding strength of a protein to the ion exchanger but describe the degree of its ionization as a function of pH [14]. Strong ion exchangers are completely ionized over a wide pH range, while weak ion exchangers are only partially ionized a narrow pH range [1,11]. Therefore with strong ion exchangers proteins can adsorb to several exchanger sites. For this reason strong ion exchangers are generally used for initial development and optimization of purification protocols. On the other hand weak ion exchangers are more flexible in terms of selectivi‐ ty and are a more general option for the separation of proteins that retain their functional‐ ity over the pH 6-9 range as well as for unstable proteins that may require mild elution conditions [11]. Alkylated amino groups for anion exchangers and carboxy, sulfo as well as phosphato groups for cation exchangers are the most common functional groups used on ion exchange chromatography supports [14]. Sulfonic acid exchangers are known as strong acid type cation exchangers. Quaternary amine functional groups are the strong base exchangers whereas less substituted amines known as weak base exchangers [1]. Number and kind of the substituents are determined the basicity of amino-groups. Immobilized tertiary and quaternary amines proved to be useful for ion exchange chromatography. Immobilized diethylaminoethyl and carboxymethyl groups are the most widely used ion exchangers [11].

**•** Dextran; Considerable swelling as a function of ionic milieu, improved materials by cross-

**•** Agarose; Swelling is almost independent of ionic strength and pH, high binding capacity

**•** Polystyrene-divinilybenzene; Hydrophobic surface, low binding capacity for proteins

In addition to electrostatic interactions between stationary phase and proteins, some further mechanisms such as hydrophobic interactions, hydrogen bonding may contribute to protein binding. Hydrophobic interactions especially occur with synthetic resin ion exchangers such as which are produced by copolymerization of styrene and divinylbenzene. These materials are not usually used for separation of proteins. However new ion exchange materials that consist of styrene-divinylbenzene copolymer beads coated with hydrophilic ion exchanger film were introduced. According to the retention behavior of some proteins, it is considered that coating of the beads so efficient that unspecific binding due to hydrophobic interactions cannot be observed. Silica particles have also been coated with hydrophilic matrix. Acrylic acid polymers are also used for the protein separation in ion exchange chromatography. These

The functional groups substituted onto a chromatographic matrix determine the charge of an ion exchange medium; positively-charged anion exchanger or a negatively-charged cation exchanger [13]. Both exchangers can be further classified as strong and weak type as shown in Table 1. The terms weak and strong are not related to the binding strength of a protein to the ion exchanger but describe the degree of its ionization as a function of pH [14]. Strong ion exchangers are completely ionized over a wide pH range, while weak ion exchangers are only partially ionized a narrow pH range [1,11]. Therefore with strong ion exchangers proteins can adsorb to several exchanger sites. For this reason strong ion exchangers are generally used for initial development and optimization of purification protocols. On the other hand weak ion exchangers are more flexible in terms of selectivi‐ ty and are a more general option for the separation of proteins that retain their functional‐ ity over the pH 6-9 range as well as for unstable proteins that may require mild elution conditions [11]. Alkylated amino groups for anion exchangers and carboxy, sulfo as well as phosphato groups for cation exchangers are the most common functional groups used on ion exchange chromatography supports [14]. Sulfonic acid exchangers are known as strong acid type cation exchangers. Quaternary amine functional groups are the strong base exchangers whereas less substituted amines known as weak base exchangers [1]. Number and kind of the substituents are determined the basicity of amino-groups. Immobilized

obtained by production of highly porous particles

**•** Polyacrylamide; Swelling behavior similar to dextran

**•** Coated polystyrene-divinilybenzene; Hydrophilic surface

polymers are especially suitable for purification of basic proteins [14].

**•** Acrylate-copolymer; High pH stability

**•** Silica; Unstable at pH > 8, rigid particles

**•** Coated Silica; Hydrophilic surface [14]

linking)

42 Column Chromatography

Theionexchangecapacityofanion-exchangerisdeterminedbythenumberoffunctionalgroups per unit weight of the resin [13]. The total ionic capacity is the number of charged functional groupspermlmedium, a fixedparameterof eachmediumandcanbegivenasmval/mlfor small ions. Density and accessibility of these charged groups both on the surface and within the pores define the total binding capacity. Ionic medium and the presence of other proteins if a particu‐ lar protein is considered also affect the binding capacity. However, under defined conditions, the amount of the certain protein which is bound to ion exchanger is more suitable parameter for determining and comparing the capacity of ion exchange chromatography. Albumin for anion exchangers and hemoglobin for cation exchangers is usually used for this purpose. Determinationofthebindingcapacitybeforetheexperimentisgenerallyrecommendedbecause the capacity for aparticularproteindepends on its size andalso on the sample composition.The binding capacity of a column can be increased for proteins which are retained on the column at high salt concentrations. The salt concentration is adjusted to a suitable concentration in which the protein of interest tightly bound to the ion exchanger while others which have lower affinity pass through the column without occupying binding sites [14].


**Table 1.** Weak and Strong type anion and cation exchangers

### **1.3. Mobile phase (Eluent)**

In ion exchange chromatography generally eluents which consist of an aqueous solution of a suitable salt or mixtures of salts with a small percentage of an organic solvent are used in which most of the ionic compounds are dissolved better than in others in. Therefore the application of various samples is much easier [1,3]. Sodium chloride is probably the most widely used and mild eluent for protein separation due to has no important effect on protein structure. However NaCl is not always the best eluent for protein separation. Retention times, peak widths of eluted protein, so chromatographic resolution are affected by the nature of anions and cations used. These effects can be observed more clearly with anion exchangers as compared to cation exchangers [14]. The salt mixture can itself be a buffer or a separate buffer can be added to the eluent if required. The competing ion which has the function of eluting sample components through the column within reasonable time is the essential component of eluting sample. Nature and concentration of the competing ions and pH of the eluent are the most important properties affecting the elution character‐ istics of solute ions [1].

to keep exposure of eluent to air to a minimum after preparation. Modern inline degas‐

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For separation the eluent is pumped through the system until equilibrium is reached, as evidenced by a stable baseline. The time required for equilibrium may vary from a couple of minutes to an hour or longer, depending on the type of resin and eluent used [10]. Before the sample injection to the column should be equilibrated with eluent to cover all the exchange sites on the stationary phase with the same counter ion. When the column is equilibrated with a solution of competing ion, counter ions associated with the fixed ions being completely replaced with competing ions. In this condition the competing ions become the new counter ions at the ion exchange sites and the column is in the form of

Isocratic elution or gradient elution can be applied for elution procedure. A single buffer is used throughout the entire separation in isocratic elution. Sample components are loosely adsorbed to the column matrix. As each protein will have different distribution coeffi‐ cient separation will achieved by its relative speeds of migration over the column. Therefore in order to obtain optimum resolution of sample components, a small sample volume and long exchanger column are necessary. This technique is time consuming and the desired protein invariably elutes in a large volume. However no gradient-forming apparatus is required and the column regeneration is needless. Alteration in the eluent composition is needed to achieve desorption of desired protein completely. To promote desired protein desorption continuous or stepwise variations in the ionic strength and/or pH of the eluent are provided with gradient elution. Continuous gradients generally give

Additives which are protective agents found in the mobile phase are generally used for maintain structure and function of the proteins to be purified. This is achieved by stimulating an adequate microenvironment protection against oxidation or against enzymatic attacks [14]. Any additives used in ion exchange chromatography, should be checked for their charge properties at the working pH in order to avoid undesired effects due to adsorption and desorption processes during chromatography [13-14]. It is recom‐ mended to include in the elution buffer those additives in a suitable concentration which have been used for stabilization and solubilization of the sample. Otherwise precipitation may occur on the column during elution [14]. For example; zwitterionic additives such as betaine can prevent precipitation and can be used at high concentrations without interfer‐ ing with the gradient elution. Detergents are generally useful for solubilization of pro‐ teins with low aqueous solubility. Anionic, cationic, zwitterionic and non-ionic (neutral) detergents can be used during ion exchange chromatography. Guanidine hydrochloride or urea, known as denaturing agents can be used for initial solubilization of a sample and during separation. However, they should use if there is a requirement. Guanidine is a charged molecule and therefore can participate to the ion exchange process in the same

sers are becoming quite popular [10].

better resolution than stepwise gradients [11].

way as NaCl during separation process [13].

that particular ion [1].

The eluent pH has considerable effects on the functional group which exist on the ion exchange matrix and also on the forms of both eluent and solute ions. The selectivity coefficient existing between the competing ion and a particular solute ion will determine the degree of that which competing ion can displace the solute ion from the stationary phase. As different competing ions will have different selectivity coefficients, it follows that the nature of competing ion will be an important factor in determining whether solute ions will be eluted readily. The concentration of competing ion exerts a significant effect by influencing the position of the equilibrium point for ion-exchange equilibrium. The higher concentration of the competing ion in the eluent is more effectively displace solute ions from the stationary phase, therefore solute is eluted more rapidly from the column. Additionally elution of the solute is influenced by the eluent flow-rate and the tempera‐ ture. Faster flow rates cause to lower elution volumes because the solute ions have less opportunity to interact with the fixed ions. Temperature has relatively less impact, which can be change according to ion exchange material type. Enhancement of the temperature increases the rate of diffusion within the ion-exchange matrix, generally leading to increased interaction with the fixed ions and therefore larger elution volumes. At higher temperatures chromatographic efficiency is usually improved [1].

Eluent degassing is important due to trap in the check valve causing the prime loose of pump. Loss of prime results in erratic eluent flow or no flow at all. Sometimes only one pump head will lose its prime and the pressure will fluctuate in rhythm with the pump stroke. Another reason for removing dissolved air from the eluent is because air can get result in changes in the effective concentration of the eluent. Carbon dioxide from air dissolved in water forms of carbonic acid. Carbonic acid can change the effective concen‐ tration of a basic eluent including solutions of sodium hydroxide, bicarbonate and carbonate. Usually degassed water is used to prepare eluents and efforts should be made to keep exposure of eluent to air to a minimum after preparation. Modern inline degas‐ sers are becoming quite popular [10].

**1.3. Mobile phase (Eluent)**

44 Column Chromatography

istics of solute ions [1].

In ion exchange chromatography generally eluents which consist of an aqueous solution of a suitable salt or mixtures of salts with a small percentage of an organic solvent are used in which most of the ionic compounds are dissolved better than in others in. Therefore the application of various samples is much easier [1,3]. Sodium chloride is probably the most widely used and mild eluent for protein separation due to has no important effect on protein structure. However NaCl is not always the best eluent for protein separation. Retention times, peak widths of eluted protein, so chromatographic resolution are affected by the nature of anions and cations used. These effects can be observed more clearly with anion exchangers as compared to cation exchangers [14]. The salt mixture can itself be a buffer or a separate buffer can be added to the eluent if required. The competing ion which has the function of eluting sample components through the column within reasonable time is the essential component of eluting sample. Nature and concentration of the competing ions and pH of the eluent are the most important properties affecting the elution character‐

The eluent pH has considerable effects on the functional group which exist on the ion exchange matrix and also on the forms of both eluent and solute ions. The selectivity coefficient existing between the competing ion and a particular solute ion will determine the degree of that which competing ion can displace the solute ion from the stationary phase. As different competing ions will have different selectivity coefficients, it follows that the nature of competing ion will be an important factor in determining whether solute ions will be eluted readily. The concentration of competing ion exerts a significant effect by influencing the position of the equilibrium point for ion-exchange equilibrium. The higher concentration of the competing ion in the eluent is more effectively displace solute ions from the stationary phase, therefore solute is eluted more rapidly from the column. Additionally elution of the solute is influenced by the eluent flow-rate and the tempera‐ ture. Faster flow rates cause to lower elution volumes because the solute ions have less opportunity to interact with the fixed ions. Temperature has relatively less impact, which can be change according to ion exchange material type. Enhancement of the temperature increases the rate of diffusion within the ion-exchange matrix, generally leading to increased interaction with the fixed ions and therefore larger elution volumes. At higher

Eluent degassing is important due to trap in the check valve causing the prime loose of pump. Loss of prime results in erratic eluent flow or no flow at all. Sometimes only one pump head will lose its prime and the pressure will fluctuate in rhythm with the pump stroke. Another reason for removing dissolved air from the eluent is because air can get result in changes in the effective concentration of the eluent. Carbon dioxide from air dissolved in water forms of carbonic acid. Carbonic acid can change the effective concen‐ tration of a basic eluent including solutions of sodium hydroxide, bicarbonate and carbonate. Usually degassed water is used to prepare eluents and efforts should be made

temperatures chromatographic efficiency is usually improved [1].

For separation the eluent is pumped through the system until equilibrium is reached, as evidenced by a stable baseline. The time required for equilibrium may vary from a couple of minutes to an hour or longer, depending on the type of resin and eluent used [10]. Before the sample injection to the column should be equilibrated with eluent to cover all the exchange sites on the stationary phase with the same counter ion. When the column is equilibrated with a solution of competing ion, counter ions associated with the fixed ions being completely replaced with competing ions. In this condition the competing ions become the new counter ions at the ion exchange sites and the column is in the form of that particular ion [1].

Isocratic elution or gradient elution can be applied for elution procedure. A single buffer is used throughout the entire separation in isocratic elution. Sample components are loosely adsorbed to the column matrix. As each protein will have different distribution coeffi‐ cient separation will achieved by its relative speeds of migration over the column. Therefore in order to obtain optimum resolution of sample components, a small sample volume and long exchanger column are necessary. This technique is time consuming and the desired protein invariably elutes in a large volume. However no gradient-forming apparatus is required and the column regeneration is needless. Alteration in the eluent composition is needed to achieve desorption of desired protein completely. To promote desired protein desorption continuous or stepwise variations in the ionic strength and/or pH of the eluent are provided with gradient elution. Continuous gradients generally give better resolution than stepwise gradients [11].

Additives which are protective agents found in the mobile phase are generally used for maintain structure and function of the proteins to be purified. This is achieved by stimulating an adequate microenvironment protection against oxidation or against enzymatic attacks [14]. Any additives used in ion exchange chromatography, should be checked for their charge properties at the working pH in order to avoid undesired effects due to adsorption and desorption processes during chromatography [13-14]. It is recom‐ mended to include in the elution buffer those additives in a suitable concentration which have been used for stabilization and solubilization of the sample. Otherwise precipitation may occur on the column during elution [14]. For example; zwitterionic additives such as betaine can prevent precipitation and can be used at high concentrations without interfer‐ ing with the gradient elution. Detergents are generally useful for solubilization of pro‐ teins with low aqueous solubility. Anionic, cationic, zwitterionic and non-ionic (neutral) detergents can be used during ion exchange chromatography. Guanidine hydrochloride or urea, known as denaturing agents can be used for initial solubilization of a sample and during separation. However, they should use if there is a requirement. Guanidine is a charged molecule and therefore can participate to the ion exchange process in the same way as NaCl during separation process [13].

Commonly used eluent additives which have been successfully used in ion exchange chro‐ matography can be given as follow;

enzymatic assays used for screening and analysis of chromatography fractions [14]. The concentration of buffer salts usually ranges from 10 to 50 mM. Commonly used buffers are

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Generally, applications of ıon exchange chromatography are performed under slightly acidic or alkali conditions, pH range 6.0-8.5 but there are also more acidic and more alkali buffers. Additionally the buffering component should not act as an eluting ion by binding to the ion exchanger. Anionic buffer component such as phosphate or MOPS in cation exchange chromatography and cationic buffers such as ethanolamine, Tris and Tricine in anion exchange chromatography are recommended. Besides interactions of buffer compo‐ nent with stationary phase, there are also possible interactions with additives of the mobile phase. To achieve sufficient buffer capacity the pKa of the buffer component should be as close to the desired pH value as possible difference no more than ± 0.5 pH units. Howev‐ er there are examples of successful separations at which the buffering capacity is very low [17-18]. It has to be considered that the pKa is a temperature dependent value. Perform‐ ing on ion exchange separation with the same elution buffer at room temperature or in the cold room can have a remarkable effect on the buffer capacity. For optimal binding of a sample ion to an ion-exchanger the ionic strength and thus also the buffer concentra‐

presented in Table 2 and Table 3 for cation and anion exchange chromatography [17].

**Substance pKa Working pH**

Citric acid 3.1 2.6-3.6

Lactic acid 3.8 3.4-4.3

Acetic acid 4.74 4.3-5.2

2-(*N*-morpholino)ethanesulfonic acid 6.1 5.6-6.6

*N*-(2-acetamido)-2-iminodiacetic acid 6.6 6.1-7.1

3-(*N*-morpholino)propanesulfonic acid 7.2 6.7-7.7

Phosphate 7.2 6.8-7.6

*N*-(2-hydroxyethyl)piperazine-*N*'-(2-ethanesulfonic acid) 7.5 7.0-8.0

N,N-bis(2-hydroxyethyl)glycine 8.3 7.6-9.0

**Table 2.** Commonly used buffers for cation-exchange chromatography

tions has to be low in sample and equilibration buffers [18].


### **1.4. Buffer**

In ion exchange chromatography, pH value is an important parameter for separation and can be controlled and adjusted carefully by means of buffer substances [18]. In order to prevent variation in matrix and protein net charge, maintenance of a constant mobile phase pH during separation is essential to avoid pH changing which can occur when both protein and exchanger ions are released into the mobile phase [11]. By means of buffer substances pH value can be controlled and adjusted. Concentration of H+ and the buffering component influence the protein binding to the stationary phase, chromatographic resolution and structural as well as functional integrity of the protein to be separated. Thus a suitable pH range, in which the stability of sample is guaranteed, has to be identified. Keeping of the sample function is related with the preservation of its three dimensional structure as well as with its biological activity [18]. A number of buffers are suitable for ion-exchange chromatography. A number of important factors influences the selection of mobile phase including buffer charge, buffer strength and buffer pH [11]. Properties of good buffers are high buffering capacity at the working pH, high solubility, high purity and low cost. The buffer salt should also provide a high buffering capacity without contributing much to the conductivity and should not interact with the ion exchanger functional groups as well as with media [11,17]. The buffering com‐ ponent should not interact with the ion exchanger because otherwise local pH shifts can occur during the exchange process which may interfere the elution. Interactions with stationary phase as well as with additives of the mobile phase and with subsequent procedures may be occur with buffer component and selected pH range. Precipitation of the mobile phase components can be observed for example when phosphate buffer and several di- and trivalent metal ions such as Mg+2 and Ca+2 are mixed or when anionic detergents (i.e. cholate) are used under acidic conditions or in the presence of multivalent metal ions. Precipitation of metal oxides and hydroxides can occur under alkaline conditions. Buffer components may also affect enzymatic assays used for screening and analysis of chromatography fractions [14]. The concentration of buffer salts usually ranges from 10 to 50 mM. Commonly used buffers are presented in Table 2 and Table 3 for cation and anion exchange chromatography [17].

Commonly used eluent additives which have been successfully used in ion exchange chro‐

In ion exchange chromatography, pH value is an important parameter for separation and can be controlled and adjusted carefully by means of buffer substances [18]. In order to prevent variation in matrix and protein net charge, maintenance of a constant mobile phase pH during separation is essential to avoid pH changing which can occur when both protein and exchanger ions are released into the mobile phase [11]. By means of buffer substances pH value can be

protein binding to the stationary phase, chromatographic resolution and structural as well as functional integrity of the protein to be separated. Thus a suitable pH range, in which the stability of sample is guaranteed, has to be identified. Keeping of the sample function is related with the preservation of its three dimensional structure as well as with its biological activity [18]. A number of buffers are suitable for ion-exchange chromatography. A number of important factors influences the selection of mobile phase including buffer charge, buffer strength and buffer pH [11]. Properties of good buffers are high buffering capacity at the working pH, high solubility, high purity and low cost. The buffer salt should also provide a high buffering capacity without contributing much to the conductivity and should not interact with the ion exchanger functional groups as well as with media [11,17]. The buffering com‐ ponent should not interact with the ion exchanger because otherwise local pH shifts can occur during the exchange process which may interfere the elution. Interactions with stationary phase as well as with additives of the mobile phase and with subsequent procedures may be occur with buffer component and selected pH range. Precipitation of the mobile phase components can be observed for example when phosphate buffer and several di- and trivalent metal ions such as Mg+2 and Ca+2 are mixed or when anionic detergents (i.e. cholate) are used under acidic conditions or in the presence of multivalent metal ions. Precipitation of metal oxides and hydroxides can occur under alkaline conditions. Buffer components may also affect

and the buffering component influence the

matography can be given as follow;

**•** Urea and guanidinium chloride

**•** Detergents;

46 Column Chromatography

**•** Organic solvents

**•** Sulfhydryl reagents

**•** Protease inhibitors [14]

**•** Zwitterions

**•** Ligands

**1.4. Buffer**

**•** Lipids

**•** EDTA; Ethylenediamine tetraacetic acid

**•** Polyols; Glycerol, glucose, and saccharose

controlled and adjusted. Concentration of H+

Generally, applications of ıon exchange chromatography are performed under slightly acidic or alkali conditions, pH range 6.0-8.5 but there are also more acidic and more alkali buffers. Additionally the buffering component should not act as an eluting ion by binding to the ion exchanger. Anionic buffer component such as phosphate or MOPS in cation exchange chromatography and cationic buffers such as ethanolamine, Tris and Tricine in anion exchange chromatography are recommended. Besides interactions of buffer compo‐ nent with stationary phase, there are also possible interactions with additives of the mobile phase. To achieve sufficient buffer capacity the pKa of the buffer component should be as close to the desired pH value as possible difference no more than ± 0.5 pH units. Howev‐ er there are examples of successful separations at which the buffering capacity is very low [17-18]. It has to be considered that the pKa is a temperature dependent value. Perform‐ ing on ion exchange separation with the same elution buffer at room temperature or in the cold room can have a remarkable effect on the buffer capacity. For optimal binding of a sample ion to an ion-exchanger the ionic strength and thus also the buffer concentra‐ tions has to be low in sample and equilibration buffers [18].


**Table 2.** Commonly used buffers for cation-exchange chromatography


**2. Ion exchange chromatograpy applications**

**Source:** *Nigella sativa* Linn.

**Detection:** UV detector at 280 nm

min. **Detection:** Pulsed amperometric detection

stachyose [20].

**Source:** *Olea europea* L.

**Sample 1:**

**Sample 2:**

**Sample 3:**

**Source:** Soybean

Ion exchange chromatography can be applied for the separation and purification of many charged or ionizable molecules such as proteins, peptides, enzymes, nucleotides, DNA, antibiotics, vitamins and etc. from natural sources or synthetic origin. Examples in which ion exchange chromatography was used as a liquid chromatograpic technique for separation or

**Extraction procedure:** Water extract of *N. sativa* was prepared, dried and powdered. Powder was dissolved in

using 3500 MW cut off dialyzing bags and centrifuged.

**Extraction procedure:** Extract was prepared from the leaves and roots of two years old olive plants with water

**Eluent:** 0.05 M phosphate buffer (pH 6.4) containing 0.01 M NaCl. Fractions of each were collected with an increasing concentration of NaCl

**Analyte(s):** Number of protein bands ranging from 94-10 kDa molecular mass [19].

(Dionex) to remove anion contaminants. **Stationary Phase:** Two anion exchange columns Dionex CarboPac PA1 plus a guard column and

Performance Anion Exchange Chromatography). **Eluent:** Eluent was comprised 12 mM NaOH with 1 mM barium acetate. Flow rate was 1 mL/

**Analyte(s):** *myo*-inositol, galactinol, mannitol, galactose, glucose, fructose, sucrose, raffinose and

**Extraction procedure:** Soybeans were defatted with petroleum ether for 30 min and centrifuged repeating

phosphate buffer saline (pH 6.4) and centrifuged at 10.000 rpm for 30 min at 4 ºC. The supernatant was collected as the soluble extract by removing the oily layer and unsoluble pellet. Protein concentration of the soluble extract was determined with Bradford method. Then proteins dialyzed against 0.05 M phosphate buffer (pH 6.4)

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at room temperature. Internal standard as D-3-*O-*methylglucopyranose (MeGlu) was used and added in appropriate volume. Extraction was accomplished by shaking for 15 min and finally the suspension was centrifuged at 3000 rpm for 10 min. Before the injection the aqueous phase was filtered and passed on a cartridge OnGuard A

CarboPac MA1 column with a guard column were used for separation procedure (High

the procedure twice. Then proteins were extracted with 0.03 M Tris-HCL buffer containing 0.01 M 2-mercaptoethanol (pH 8) for 1 hour following by centrifugation (16.250 x g for 20 min at 20 ºC). The supernatant was adjusted to pH 6.4 with 2 M HCl and centrifuged (16.250 x g for 20 min at 2-5 ºC). The precipitate was dissolved in Tris-HCl buffer and the process was repeated in order to obtain purified precipitated fraction containing the 11S globulin. The supernatant obtained after the first

purification of bioactive molecules from natural sources can be given as below.

**Stationary Phase:** XK50/30 column (5 x 15 cm) of DEAE sephadex A50.

**Table 3.** Commonly used buffers for anion-exchange chromatography
