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Catalog Record: Ion exchange in analytical chemistry | HathiTrust Digital Library
RSS Feeds. Equilibrium is established for each sample component between the eluent and stationary phases when a sample is introduced into the ion-exchange chromatography. The value of D A 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:. V m : 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 ].
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.
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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;.
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-exchange matrix itself [ 1 ].
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 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 broadening.
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 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 ].
Schematic presentation of different matrix types a non-porous beads b microporous beads c macroporous 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 conventional 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 chromatographic resolution. Stationary phases with particle of uniform size are superior to heterogenous 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 In order to minimize non-specific interactions with sample components inert matrix should be used.
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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 backbones are more suitable matrices for protein separations.
Other ion exchange matrices with hydrophilic properties are based on agarose or dextran [ 14 ]. Dextran; Considerable swelling as a function of ionic milieu, improved materials by cross-linking. Agarose; Swelling is almost independent of ionic strength and pH, high binding capacity obtained by production of highly porous particles. Coated Silica; Hydrophilic surface [ 14 ]. 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.https://tr.debecyfekeko.tk
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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 ].
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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.
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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 selectivity and are a more general option for the separation of proteins that retain their functionality over the pH 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 ].